Pathology Research 2013

PDF (2.1 MB)

A.

Fixatives

A-1. for Light Microscopy

A-2. for Electron Microscopy

B.

Tissue Dehydrating Solution

 

C.

Decalcifying Agent

 

D.

Intermediates (Replacing agents)

D-1. Lemosol® and Lemosol® A (Ace)

E.

Embedding Medium

 

F.

Immunopen

 

G.

Stains

G-1. General Staining

G-2. Polysaccharide Staining

G-3. Connective Tissue Staining

G-4. Staining of Fibrins and Glial Tissues

G-5. Intracellular Inorganic Substance Staining

G-6. Cytological Staining

G-7. Blood Cell Staining

G-8. Hard Tissue Staining (TRAP/ALP Stain Kit)

G-9. Other stains

H.

Mounting Reagent

H-1. Softmount

I.

Antigen Retrieval Reagent

I-1. ImmunoSaver

J.

Gene-Related Kits

J-1. Apoptosis in situ Detection Kit Wako

J-2. DNA Isolator PS Kit

J-3. DNA Isolator PS-Rapid Reagent

J-4. ISOGEN PB Kit

K.

Paraffin-embedded Tissue Sections

K-1. HistoMap Series

A.

Fixatives

Fixation means a process by which when observing microscopically the ecological or pathological state of a tissue specimen taken from a dead or live body, the tissue specimen is preserved in as "intact" a state as possible.

The objectives are to:

A-1.

for Light Microscopy

Neutral buffered formalin

Formalin solution is used in large quantities as a routine fixative in the preparation of tissue specimens for light microscopic observation. Formalin is decomposed to produce formic acid, which may affect some tissues. This neutral buffered formalin is adjusted to pH 7.4 approximately by the addition of sodium phosphate to formalin to eliminate the effect of acid on tissues.

Not only is neutral buffered formalin a superior fixative for routine lab work, it is also beneficial for a number of special purposes. For example, neutral buffered formalin can be used to a certain extent for histochemical detection of enzymes, hemoglobin and other molecules; for fixing mucopolysaccharides, glycogens, neurofibrils and other tissue components that cannot be treated with regular formalin; and for electron microscopy.

Product Name

Composition

Formaldehyde

Content

pH

Penetration Strength

10% Formalin

Neutral Buffer Solution

Formalin ……100 mL

Sodium phosphate, monobasic, Dihydrate ……4.5 g

Disodium phosphate ……6.5 g

Prepare the 1 L solution to add ion-exchanged water

3.7~4.3 w/w%

7.4~7.5

  • Penetration strength is in the order of 20% > 15% > 10%.
  • Specimens must be placed in the 10% fixative for several days or more.
  • The 20% fixative allows fixation in one or two days.
  • The 15% solution is about halfway between the 10% and 20% solutions.

15% Formalin

Neutral Buffer Solution

Formalin ……150 mL

Sodium phosphate, monobasic, Dihydrate ……4.5 g

Disodium phosphate ……6.5 g

Prepare the 1 L solution to add ion-exchanged water

5.6~6.4 w/w%

7.35~7.45

20% Formalin

Neutral Buffer Solution

Formalin ……200 mL

Sodium phosphate, monobasic, Dihydrate 4.5 g

Disodium phosphate ……6.5 g

Prepare the 1 L solution to add ion-exchanged water

7.5~8.5 w/w%

7.3~7.5

Product Name

Grade

Package Size

Wako Cat. No.

10% Formalin Neutral Buffer Solution

for Tissue Fixation

1 L

062-01661

20 L

060-01667

15% Formalin Neutral Buffer Solution

for Tissue Fixation

1 L

069-02391

20 L

067-02397

20% Formalin Neutral Buffer Solution

for Tissue Fixation

1 L

060-01721

20 L

068-01727

Mildform

Mildform products, fixatives for pathological tissues, are neutral buffered formalin solution prepared according to Lillie’s formulation to which wine extract* is added. The wine extract minimizes the irritating and unpleasant odor of formalin, however, with regard to operational safety, minimum degree of odor is present so that formalin can be recognized.

* Mechanism of wine extract’s effect: The masking effect minimizes the irritating and unpleasant odor of formalin.

Features

Data

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Product Name

Grade

Package Size

Wako Cat. No.

Mildform 10N

for Pathology Research

1 L

133-10311

20 L

131-10317

Mildform 15N

for Pathology Research

1 L

132-14301

20 L

130-14307

Mildform 20N

for Pathology Research

1 L

136-10041

20 L

134-10047

Mildform 10NM

for Pathology Research

1 L

132-10521

20 L

130-10527

Mildform 15NM

for Pathology Research

1 L

139-14311

20 L

137-14317

Mildform 20NM

for Pathology Research

1 L

139-10531

20 L

137-10537

<Note>
Although Mildform lacks the unpleasant, acerbic odor of formalin, it is still as toxic as regular formalin and therefore should be handled with due care.

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Other Fixatives

Product Name

Composition

Features

Bouin Solution

Saturated Picric Acid Solution ……750 mL

Formalin Stock Solution ……250 mL

Acetic Acid, Glacial ……50 mL

This product allows fixation of small sections in 1 to 2 hours because of its strong penetration. Fetal bone tissue can be thinly sliced because the solution’s decalcifying effect is relatively weak. This fixative is used frequently for endocrine tissue, glycogens and polysaccharides. After fixation, wash specimen with 70% ethanol until the yellow color disappears.

Carnoy Solution

Pure Ethanol ……600 mL

Chloroform ……300 mL

Acetic Acid, Glacial ……100 mL

With better penetration performance than anhydrous alcohol, this product is suited for the fixation of glycogens and nucleic acids. It can fix 3 to 4 mm specimens in a matter of 2 to 4 hours. After fixation, transfer the specimen to pure alcohol to remove chloroform and acetic acid, and then embed. Because this product has strong dehydrating and delipidating properties, overfixation will lead to extreme contraction and hardening of the specimen.

4% Paraformaldehyde

Phosphate Buffer Solution

Paraformaldehyde ……20 g

0.1 mol/L Phosphate buffer (pH 7.4) ……500 mL

The product is used in enzyme histochemistry and immunohistochemistry, and as a fixing solution for electron microscopy.

It is used for fixing protein antigens in immunostaining.

PLP Solution Set

Solution A

(Lysine-phosphate buffer solution) ……50 mL

Solution B

(10 % Paraformaldehyde solution) ……50 mL

Solution C

(Metaperiodic acid solution) ……50 mL

This neutral buffered fixative is specially made for immunohistochemistry and is suited for fixing glycoprotein antigens. It is used to detect surface makers of complements and lymphocytes that are prone to losing their antigenicity.

Zamboni Solution

Saturated Picric Acid Solution ……150 mL

Paraformaldehyde ……20 g

Add 320mOSM Phosphate buffer to make the 1 L solution

This fixative is used primarily for immunohistochemistry and electron microscopy, and is suited for fixing polypeptide antigens. Fixation takes about 4 to 12 hours at 4°C. After fixation, wash specimen with 70% ethanol until the yellow color disappears.

Data

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Product Name

Grade

Package Size

Wako Cat. No.

Bouin Solution

for Pathology Research

1 L

023-17361

Carnoy Solution

for Tissue Fixation

1 L

034-17711

4% Paraformaldehyde Phosphate Buffer Solution

for Tissue Fixation

100 mL

161-20141

500 mL

163-20145

PLP Solution Set

for Tissue Fixation

for 250 mL

290-63201

Zamboni Solution

for Pathology Research

1 L

263-01991

 

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A-2.

for Electron Microscopy

Fixatives for Electron Microscopy

Fixatives

Working

Concentration

Advantage

Disadvantage

Applications

Formalin Neutral Buffer

4%

Can effectively fix soluble proteins (e.g. functional proteins such as hormones and enzymes) and polypeptides

-

Soluble protein, polypeptide

Glutaraldehyde

1.5~4%

Enables negative imaging of intracellular structure, glycogen granules; Enables positive imaging of nuclear and intracellular matrices

More time required for washing and dehydrating; weak penetration of embedding materials other than methacrylic resin; inadequate fixation of lipids; no fixation of proteins at low concentrations

Polysaccharides

Osmic Acid

1~2%

Reduces washing and dehydration time; less deformation during fixing; good embedding agent penetration enables use of various types of resin; ideal for fixing phospholipids

Weak penetration; causes loss saturated fatty acids and 50% of all proteins

Intracellular microstructures; unsaturated fatty acids; DNA granules

Paraformaldehyde

4%

Can be used for fixing glycoprotein and preserving antigenicity; penetrates tissue rapidly

Weak fixation

Protein antigens

PLP

(Periodate-lysine-PFA)

2%

Suitable for fixing glycoprotein and sugar; Can keep antigenicity

-

Glycoprotein antigens

Potassium

permanganate

0.6~3%

As a fixative, exhibits medium penetration of the embedding material

Cases much deformation and partial loss of mucopolysaccharides, proteins, saturated fatty acids and phospholipids; does not fix ribosomes

Intracellular microstructures; glycogen granules; plant specimens

Tannic Acid, Aldehyde

1%, 2%

Can effectively fix soluble proteins (e.g. functional proteins such as hormones and enzymes) and polypeptides when mixed with an aldehydes fixative

-

Soluble protein, polypeptide

Product Name

Grade

Package Size

Wako Cat. No.

10% Glutaraldehyde Solution

for Electron Microscopy

2 mL × 5

071-02031

20% Glutaraldehyde Solution

for Electron Microscopy

25 mL

072-02262

500 mL

076-02265

70% Glutaraldehyde Solution

for Electron Microscopy

2 mL × 5

071-01931

Osmium (VIII) Oxide

for Electron Microscopy

1 g

154-01014

2% Osmium (VIII) Oxide Solution

for Electron Microscopy

5 mL × 5

154-01151

4% Osmium (VIII) Oxide Solution

for Electron Microscopy

5 mL × 5

157-01141

Paraformaldehyde

for Tissue Fixation

100 g

160-16061

500 g

162-16065

2,4,6-Trimethylpyridine [γ-Collidine]

for Electron Microscopy

25 mL

035-08093

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B

Tissue Dehydrating Solution

B.

Tissue Dehydrating Solution

In the course of preparing pathology specimens, tissues are thinly sliced after paraffinization and embedding block assembly. In the same manner that water and oil do not mix, paraffin, an oil derivative, will not penetrate wet tissue samples. Therefore, it is necessary to dehydrate the specimen with alcohol and then replace the alcohol with an intermediate. Consequently, alcohol is an indispensable solvent for preparing pathology specimens. However, after amendment of the Liquor Tax Act in 1994 resulted in higher ethanol prices researchers began looking for a cheaper alternative with the same properties. Our tissue dehydrating solutions are made of denatured alcohol with reduced water content and provide the same performance as ethanol. Moreover, these products are affordable because they do not fall within the purview of the Liquor Tax Act. Product specifications are listed below.

 

for Electron Microscopy

 

Tissue Dehydration Solution 99

Tissue Dehydration Solution 100

Component

Ethanol: 99+%

Acetone: 0.7%

Dehydration Agent: Zeolite 3Å type abt. 3g packed in a bag

Denaturant: containing Bitrex™: 10 ppm

Ethanol: 99.8+%

Dehydration Agent: Zeolite 3Å type abt. 3g packed in a bag

Denaturant: containing Bitrex™: 10 ppm

Water

≤ 0.2%

≤ 0.2%

Feature

the same purity and performance of conventional ethanol

Product Name

Grade

Package Size

Wako Cat. No.

Tissue Dehydration Solution 99

for Electron Microscopy

500 mL

205-17445

Tissue Dehydration Solution 100

for Electron Microscopy

500 mL

208-17435

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C.

Decalcifying Agent

Using microtome blades to prepare thin sections of hard tissue such as bone and calcified foci is a formidable challenge. Decalcifying the specimen in advance makes the process easier. Thin slicing can be done more efficiently if the calcium carbonate in the tissue is dissolved with acid or some other solvent and converted into a softer salt. A proper decalcifier needs to possess the following properties.

Basic Protocol

Resection Dehydrate/Delipidate Hydrate Wash Decalcify Neutralize Dehydrate Remove alcohol Paraffinize Slice Deparaffinize Stain

 

Kalkitox™

This product is a new type of decalcifier that combines the advantages of the Plank-Rychlo and chelating agent methods.

Instructions

Decalcification takes about one night at room temperature, although this will depend to a certain extent on the size of the specimen. With Kalkitox, we recommend low-temperature decalcification (refrigeration at 4°C for one or two nights), followed by neutralization in sodium sulfate solution for several hours to one night, and then thorough washing with water. Afterwards, perform dehydration, solution replacement with an intermediate and then paraffinization.

Advantages

Composition

Main ingredients are hydrochloric acid and EDTA.

Data

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Product Name

Grade

Package Size

Wako Cat. No.

Kalkitox™

for Pathology Research

1 L

112-00651

Decalcifying Solution A, B

Decalcification

Decalcifying Solution

Composition

Features

Rapid Decalcification

Decalcifying Solution A

(Plank-Rychlo protocol)

· Aluminum Chloride ……7.0 g

· Hydrochloric Acid ……8.5 mL

· Formic Acid ……5.0 mL

Prepare the 100 mL solution with distilled water

· Processing time is about 3x the length for formic or hydrochloric acid

· Poor maintenance of antigenicity

· Requires neutralization with 5% sodium sulfate

Neutral Decalcification

Decalcifying Solution B

(EDTA protocol)

Main component: 0.5 mol/L EDTA solution (pH 7.0 ~ 7.5)

· Less damage to tissue

· Good preservation of structure and staining

· Ideal for immunohistostaining

· Decalcification requires a longer length of time

Data

 

Decalcifying Solution A

Decalcifying Solution B

He Staining

 

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Complete decalcification after 12 hours

bone thickness of 10 mm

Fast decalcification

Complete decalcification after 5 days

bone thickness of 3 mm

Slow decalcification

Immunohistostaining

 

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Complete decalcification after 12 hours

bone thickness of 3 mm

Loss of carcinoembryonic antigen sites

Complete decalcification after 5 days

bone thickness of 3 mm

Carcinoembryonic antigen sites are kept.

Product Name

Grade

Package Size

Wako Cat. No.

Decalcifying Solution A (Plank-Rychlo protocol)

for Pathology Research

1 L

047-21911

Decalcifying Solution B (EDTA protocol)

for Pathology Research

1 L

047-21911

Morse Solution

Sections of bone tissues, calcification foci, and calcium deposited lesions cannot be prepared as they are. Therefore, they need decalcification. In this procedure, tissue calcium carbonate is degraded to soft calcium salts with an acid allowing easy sectioning. This product decalcifies 8-time more quickly than neutral EDTA solution and the results are said to be comparable to EDTA in immunostaining and in situ hybridization.

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Composition

· 22.5% Formic Acid

· 10% Sodium Citrate

Rat nasal cavity

HE Staining

Product Name

Grade

Package Size

Wako Cat. No.

Morse Solution

for Pathology Research

1 L

135-17071

<Related Products>

Product Name

Grade

Package Size

Wako Cat. No.

5% Formic Acid (for Decalcification)

for Pathology Research

500 mL

062-04025

10% Hydrochloric Acid (for Decalcification)

for Pathology Research

500 mL

089-07675

8% Nitric Acid (for Decalcification)

for Pathology Research

500 mL

146-07075

5% Sodium Sulfate Solution (for Decalcification)

for Pathology Research

500 mL

196-11985

5% Trichloroacetic Acid Solution (for Decalcification)

for Pathology Research

500 mL

205-14905

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D.

Intermediates (Replacing agents)

Although you may have replaced the water in the dehydrated tissue with alcohol, you will need to replace this alcohol with a solution compatible with paraffin. This is because alcohol and paraffin do not mix well. Currently popular intermediates include alcohol and paraffin-compatible xylene. However, xylene is highly volatile, foul-smelling and hazardous to health. These adverse properties have made use of paraffin in the workplace a controversial issue.

Lemosol® and Lemosol® A are alternatives to xylene that are virtually non-toxic and do not emit offensive odors.

Lemosol® A is particularly unique because it contains phytoncide, the substance responsible for the pleasant odor of forests.

D-1.

Lemosol®, Lemosol® A (Ace)

Decalcification

Lemosol®

Lemosol® A (Ace)

Main component

D(+)-Limonene

Pinene

Origin

orange peel

bark of eucalyptus and pine

Smell

Citrus

Eucalyptus and pine resin

Volatility

Less volatile compared to xylene

Superior volatility

Specific Gravity (20°C)

0.838 ~ 0.844 g/mL

0.873 ~ 0.879 g/mL

Flashing Point

61°C

36.5°C

Toxicity (LD50 rat p.o.)

≥ 5 g/kg

Does not irritate the eyes, nose, throat or skin.

Limonene has been approved by the U.S. Food and Drug Agency as a food additive and flavoring agent.

≥ 5 g/kg

Does not irritate the eyes, nose, throat or skin.

Pinene has been approved by the U.S. Food and Drug Agency as a food additive and flavoring agent.

Boiling Point

177°C (d-Limonene)

154~166°C

Refractive Index (20°C)

1.473

1.475

Lemosol®

Lemosol® is a safe substitute for xylene and chloroform. Its pleasant lemon fragrance comes from one of its main ingredients, D-(+)-Limonene, which is a substance derived from orange peel oil.

Features

 

Examples of stained sections prepared with Lemosol®

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Lemosol® A (Ace)

Lemosol® A is terpene-based solvent derived from bark of eucalyptus and pine.

Lemosol® A can be used in place of xylene as an intermediate and clearing agent for the preparation of pathology specimens. An advanced version of Lemosol®, this product and does not emit offensive odors and exhibits good volatility.

 

Features

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Example of a protocol employing an intermediate

70% Alcohol

for 1 hr.

70% Alcohol

for 1 hr.

70% Alcohol

for 1 hr.

70% Alcohol

for 1 hr.

100% Alcohol

I

for 1 hr.

II

for 1 hr.

III

for 1.5 hr.

IV

for 1.5 hr.

Intermediate

I

for 1 hr.

II

for 1 hr.

Paraffin

I

for 1 hr.

II

for 1 hr.

Paraffin

[Melting container at 62°C]

III

for 10 min.

IV

for 10 min.

Paraffin [vacuum]

V

for 10 min.

 

Conditions for deparaffinization and alcohol processing

Intermediate

I

for 2 ~ 3 min.

II

for 3 ~ 5 min.

III

for 3 ~ 5 min.

100% Alcohol

I

 

II

 

III

 

Wash

 

Conditions for clearing and mounting

100% Alcohol

I

 

II

 

III

 

Intermediate

I

for 1 hr.

II

for 1 hr.

III

for 1 hr.

Clear with xylene

Mount

Disposal method for Lemosol® and Lemosol® A

We can provide a coagulant for safe disposal.

Flushing solution down a drain that directly leads to public waters will result in a rise of BOD and TOC. Therefore, if you are planning to dispose of solution in its liquid form, treat beforehand with activated sludge or some other appropriate method.

Product Name

Grade

Package Size

Wako Cat. No.

Lemosol®

for Pathology Research

1 L

122-03991

3 L

128-03993

Lemosol® A (Ace)

for Pathology Research

1 L

120-04411

3 L

126-04413

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Lemosol® Coagulant

This product is a coagulant for the safe disposal of Lemosol® and Lemosol® A (Ace), which are used as intermediates or clearing agents for the preparation of pathology specimens.

 

Main Component

Hydrogenated castor oil fatty acid

 

Basic Protocol

Add an amount of Lemosol® coagulant that is approximately 5% the volume of the Lemosol® or Lemosol® A to be disposed.

Place the coagulant and solution mixture on a hot plate stirrer or in a hot water bath. Heat to about 60°C and agitate in order to melt the contents. (Be careful not to cause a fire.)

Cool the mixture until solid.

<Note>

Both Lemosol® and Lemosol® A are highly flammable. Do not expose to open flames. Moreover, both products should not be handled near any type of flame.

After coagulation, handle the generated waste as a Class II Hazardous Material No. 1 flammable solid.

Waste solution containing xylene (regulated as a deleterious material for non-medical use and as a hazardous substance under the Fire Service Act) or chloroform (regulated as a deleterious material for medical use) cannot be disposed in the manner described above.

<Specific measures>

If you are planning to store the coagulated solution indoors for a period of time:

Place the coagulated solution in a vinyl bag so the contents do not leak. Seal the bag tightly at the opening and store in a location away from any open flames.

(The contents may vaporize, especially during summer. Therefore, if the bag is not properly sealed, leaking vapors may combust on contact with an open flame.)

If you are planning to dispose the coagulated solution immediately (In case of Japan):

If the amount to be disposed at a single time is less than 20 kg, we recommend you use your regular facilities to process the waste. No special permission from the local authorities is necessary.

If the amount to be disposed at a single time is from 20 kg to less than 100 kg, you need to obtain special permission from the local authorities.

Product Name

Grade

Package Size

Wako Cat. No.

Lemosol® Coagulant

for Solidification of Used Lemosol

500 g

129-04545

E.

Embedding Medium

Pathoprep® Series

Paraffin for embedding of pathologic tissue. Pellet type.

Purified paraffin with uniform carbon distribution, to which a safe and stable high-molecular polymer has been added to increase tissue permeability. This line of products is superior in terms of sliceability, extensibility and penetration.

 

Pathoprep® 546

Pathoprep® 568

Pathoprep® 580

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M.P.: 54 ~ 56°C; DMSO-free

M.P.: 56 ~ 58°C; DMSO-free

M.P.: 58 ~ 60°C; DMSO-free

Product Name

Grade

Package Size

Wako Cat. No.

Pathoprep® 546

for Pathological Tissue Embedding

2 kg × 3

167-20501

Pathoprep® 568

for Pathological Tissue Embedding

500 g × 12

162-18961

Pathoprep® 580

for Pathological Tissue Embedding

2 kg × 3

165-19551

Other Embedding Medium

Product Name

Grade

Package Size

Wako Cat. No.

Collodion (5%)

Wako 1st Grade

500 mL

032-03885

Collodion (10%)

Wako 1st Grade

500 mL

031-16425

Gelatin

Wako 1st Grade

500 g

077-03155

Embedding Medium for Electron Microscopy

Product Name

Grade

Package Size

Wako Cat. No.

Ethylene Glycol Diglycidyl Ether

Electron Microscopy

200 g

056-03841

500 g

058-03845

Epon 815

Electron Microscopy

200 g

056-02981

500 g

058-02985

Epon 174

Electron Microscopy

200 g

234-00781

500 g

236-00785

Epon 812

Electron Microscopy

200 g

237-00771

500 g

239-00775

<Related Products>

hardening Agents

Product Name

Grade

Package Size

Wako Cat. No.

Dodecenylsuccinic Anhydride [DDSA]

Electron Microscopy

200 g

040-16251

500 g

042-16255

Methyl-5-norbornene-2,3-dicarboxylic Anhydride [MNA]

Electron Microscopy

200 g

134-05951

500 g

136-05955

Nonenylsuccinic Anhydride [NSA]

Electron Microscopy

100 g

141-04541

500 g

143-04545

Accelerator

Product Name

Grade

Package Size

Wako Cat. No.

2,4,6-Tris (dimethylaminomethyl) phenol [DMP-30]

Electron Microscopy

25 mL

204-06263

Gelatin, Capsules

Product Name

Grade

Package Size

Wako Cat. No.

Gelatin, Capsules No.0

Electron Microscopy

100 ea.

074-01801

Gelatin, Capsules No.00

Electron Microscopy

100 ea.

071-01791

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F.

Immunopen

Immunopen is a felt-tipped pen type reagent and used as an adjunct to immunostaining. It is designed to minimize waste of antiserum in immunostaining techniques on sections mounted on slide glasses and to induce immunoreactions efficiently.

 

An example for use <Section on a slide glass plate>

1. Deparaffinizing

2. Wash in distilled deionized water.

3. Immunopen Treatment: Draw a circle around the section on the slide glass.

It becomes a barrier to protect unnecessary spreading of solutions for staining.

 

Direction for use

1. Used as a tool for immunostaining

2. Used in ABC staining and fluorescent antibody stanining

 

Precautions

1. Immunopen is insoluble in water.

Before use, thoroughly wipe up water around sections with filter papers on a slide glass.

2. Immunopen is soluble in oil. When tissue sections are fixed in acetone, use Immunopen after fixation and solvent removal.

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Product Name

Package Size

Wako Cat. No.

Immunopen

1 pen

299-20551

5 pens

295-20553

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G.

Stains

Purpose of stain

Stain Method

Stain Result

Wako’s Products

Page

General stains

(G-1)

Hematoxylin-eosin (HE) stain

Nuclei, cartilage, bacteria,

non-decalcified areas : Indigo blue

Cytoplasm, connective tissue, muscle

tissue, red blood cells :

pink to reddish pink

Mayer's Hematoxylin Solution

Mayer's Hematoxylin Solution (×2)

Carrazzi's Hematoxylin Solution (×2)

Eosin Alcohol Solution, acid extract

1% Eosin Y Solution

0.1% Eosin Y Ethanol Solution

14

Polysaccharides

(G-2)

Glycogen

PAS stain

PAS-positive substances :

Reddish purple

PAS-positive substances:

glycogen, proteins (e.g. mucin, basement membrane, fibrin compounds, some hormones, lipofuscin, fungi, Entamoeba histolytica)

Schiff's Reagent

Cold Schiff's Reagent

16

Acidic slime

polysaccharides

Alcian blue stain

Acid mucopolysaccharide : blue

Nuclei : red

Alcian Blue Solution, pH2.5

17

Toluidine blue stain

Acid mucopolysaccharide, mast cell granule : red to reddish purple

(metachromasy)

Nuclei, other tissue components : blue

0.05%Toluidine Blue Solution (pH 2.5)

0.05%Toluidine Blue Solution (pH 4.1)

0.05%Toluidine Blue Solution (pH 7.0)

18

Colloidal iron stain

Acid slime polysaccharide : blue

Nuclei : red

Colloidal Iron Stock Solution

19

Connective tissues

(G-3)

Collagen fibers

Elastica-van

Gieson stain

Collagenous fiber, reticular fiber,

basement membrane : red

Muscle fiber, cytoplasm : yellow

Nuclei : blackish brown

Weigert's Iron Hematoxylin Staining Set

Van Gieson Solution P

Van Gieson Solution F

19

Masson’s trichrome

stain

Collagenous fiber, reticular fiber, renal

glomerular basement membrane : blue

Nuclei : purplish black to purplish red

Cytoplasm : pale red to purplish red

Aniline Blue Solution

0.8% Orange G Solution

Ponceau Xylidine. Acid Fuchsin. Azophloxine Solution

20

Azan stain

Collagenous fiber, reticular fiber, renal

glomerular basement membrane :

bright blue

Nuclei : dark red

Fibrin : red

Aniline Blue · Orange G Solution

Azocarmine G Solution

21

Elastic fibers

Victoria blue stain

Elastomeric fiber : HBs antigen

Cartilaginous matrix : blue

Nuclei : blue

Background : pale pink

Victoria Blue Solution

22

Fibrin, glial tissues

(G-4)

Phosphotungstic

Acid Hematoxylin

(PTAH) stain

Glial fiber, fibrin, striated muscle

striations, nucleus, smooth muscle

fiber : bluish indigo

Connective tissue, nerve cell :

brownish red

Phosphotungstic Acid Hematoxylin

Solution

23

Intracellular inorganic substances

(G-5)

Iron

(hemosiderin)

Berlin blue stain

Hemosiderin : blue

Nucleus : red

Berlin Blue Staining Set

23

Cytodiagnosis

(G-6)

Papanicolaou stain

Nucleus : dark purple

Papanicolaou-Gill's Hematoxylin (Gill-5)

Stain Solution

Papanicolaou, Hematoxylin Stain

Solution

Papanicolaou EA100 Stain Solution

Papanicolaou OG100 Stain Solution

24

Hematocyte

(G-7)

Giemsa Stain

Wright Stain

Wright-Giemsa

Stain

May-Grünwald

Stain

Nuclei : reddish purple

Giemsa Stain Solution

Wright Stain Solution

May-Grünwald Stain Solution

May-Grünwald's Eosin Methylene Blue

Solution

25

Hard tissue

(G-8)

Stains for

osteoblast,

osteoclast

TRAP/ALP stain

Osteoblast : blue to brownish blue

Osteoclast : reddish purple

TRAP/ALP Stain Kit

26

Osteoid stains

Villanueva Bone Stain

Osteoid : reddish purple

-

 

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G-1.

General Staining

Hematoxylin-Eosin (HE) Stain

Hematoxylin-eosin (HE) stain is a method to stain the nucleus dark blue with hematoxylin and the cytoplasm dark and pale red with eosin, and is used for overview staining to know the entire cell and tissue structures.

 

Principle of Stains

It is considered that hematin generated by oxidizing hematoxylin forms a complex with the metallic portion of the mordant, is charged positively, and is then bound to the negatively charged phosphate group of the nucleus to produce staining. (It is also considered that the cytoplasm and connective tissue are charged positively and are bound to the negatively charged eosin: afterstaining with eosin)

45422.png 

Hematoxylin and Eoxin (HE) Staining Protocol

45453.png 

Mayer’s Hematoxylin Solution

This product is the most widely used Mayer’s hematoxylin solution. Double-strength Mayer’s hematoxylin ((×2) product) contains twice the amount of hematoxylin, enabling faster staining.

 

Composition

Hematoxylin ……1.0 g (In case of the (×2) product: 2.0 g)

Potassium Alum ……50 g

Sodium Iodate ……0.2 g (In case of the (×2) product: 0.4 g)

Chloral Hydrate ……50 g

Citric Acid ……1.0 g

Distilled Water ……1,000 mL pH 2.0 ~ 3.0 (25°C)

<Note>

If crystals of aluminum potassium sulfate 12-hydrate precipitate during preservation, use a supernatant solution.

 

45495.png

Renal biopsy (×200)

Product Name

Grade

Package Size

Wako Cat. No.

Mayer’s Hematoxylin Solution

for Pathology Research

500 mL

131-09665

Mayer’s Hematoxylin Solution (x2)

for Pathology Research

500 mL

134-13065

Carrazzi’s Hematoxylin Solution

Carazzi’s Hematoxylin is used primarily as a cytodiagnostic tool because, with the proper differentiation protocol, it can stain intranuclear structures with remarkable clarity.

Composition

Hematoxylin ……1.0 g (In case of the (×2) product: 2.0 g)

Potassium Alum ……50 g

Sodium Iodate ……0.2 g (In case of the (×2) product: 0.4 g)

Glycerin ……200 mL

Distilled Water ……800 mL

 

37922.png

Human Kidney

Product Name

Grade

Package Size

Wako Cat. No.

Carrazzi's Hematoxylin Solution

for Pathology Research

500 mL

032-14635

Carrazzi's Hematoxylin Solution (x2)

for Pathology Research

500 mL

039-17705

Eosin Alcohol Solution, Acid Extract

This product is alcohol solution of acid-extracted eosin for good contrast staining. It can be used immediately allowing clear staining of fine cellular structures.

Composition

Acid-extracted Eosin ……100 mL

95% Alcohol ……800 mL

Acetic Acid ……8 mL

Examples of staining

37981.png

Using Acid extracted Eosin

Using conventional Eosin

Product Name

Grade

Package Size

Wako Cat. No.

Eosin Alcohol Solution, acid extract

for Pathology Research

1 L

050-06041

Eosin Y Solutions

Composition

· 1% Eosin Y Solution

Eosin Y ……5.0 g

Distilled Water ……500 mL

Acetic Acid ……A few drops

· 0.1% Eosin Y Solution

10% Eosin Y Solution ……5 mL

95% Alcohol ……495 mL

· 0.5% Eosin Y Solution

10% Eosin Y Solution ……25 mL

95% Alcohol ……475 mL

 

The above mentioned three type products show excellent staining performance. The aqueous solution type in particular is also applicable to cryosection.

In general, higher concentrations are recommended for thinner sections.

Product Name

Grade

Package Size

Wako Cat. No.

1% Eosin Y Solution

for Pathology Research

500 mL

051-06515

0.1% Eosin Y Ethanol Solution

for Pathology Research

500 mL

054-06505

0.5% Eosin Y Ethanol Solution

for Pathology Research

500 mL

051-06495

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G-2.

Polysaccharide Staining

PAS (Periodic Acid Schiff) Stain

Schiff’s Reagent

A Schiff reagent is a stain reagent used frequently in the PAS (Periodic Acid Schiff) reaction and Feulgen’s reaction in the fields of histopathology, clinical pathology and hematology. As it reacts with aliphatic aldehydes sensitively and develops red to purple colors, it is applied extensively to intracellular staining, staining of polysaccharides such as neutral mucopolysaccharides, glycoproteins, etc. and staining of lipids constituting lipoproteins. There are two types of Schiff reagents according to preparation protocols. One is hot Schiff reagent which is prepared by boiling. The other is cold Schiff reagent which is prepared at room temperature. Although the preparation protocols differ, there is virtually no change in staining performance.

 

Advantages

Reaction Formula

 

38099.png
38181.png

 

Human Kidney

Composition

Fuchsin Basic ……10 g

Sodium metabisulfite ……19 g

0.5 mol/L Hydrochloric acid ……1,000 mL

 

45554.png

* : Sulfurous acid solution

· 10% sodium sulfite (or 10% sodium bisulfate); 6 mL

· 1 M hydrochloric acid: 5 mL

· Distilled water: 100 mL

 

Product Name

Grade

Package Size

Wako Cat. No.

Schiff’s Reagent

for Pathology Research

100 mL

191-08441

for Pathology Research

500 mL

193-08445

Cold Schiff’s Reagent

In comparison to hot Schiff reagent, cold Schiff reagent is prepared at room temperature and is better for staining sections with a thickness of 2 μm or less.

 

Staining results

Glycogen stains deep purplish red. Mucin stains red to purple. Fibrin stains pink. Reticular tissue and basement membrane stain purplish red.

PAS-positive microorganisms include fungi (yeast, blastomyces), bacteria (e.g. anthrax, gut flora) and Entamoeba histolytica.

 

Composition

Fuchsin Basic ……10 g

Sodium metabisulfite ……19 g

0.15 mol/L Hydrochloric acid ……1,000 mL

 

38346.png

Human Kidney

Product Name

Grade

Package Size

Wako Cat. No.

Cold Schiff’s Reagent

for Pathology Research

500 mL

039-14645

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Alcian Blue Stains

Alcian blue stains mucopolysaccharides by binding specifically to their sulfate and carboxyl groups. At pH 2.5, both the sulfate and carboxyl groups are stained, whereas at pH lower than 1.0, only the sulfate group is known to bind with alcian blue.

The alcian blue solution is used for double-staining with PAS, which makes it possible to stain neutral and acid mucopolysaccharides in different colors.

Each cell and tissue produces specific acid mucopolysaccharides, and by observing the tissue-specific acid mucopolysacchrides, it is possible to distinguish between particular characteristics of each tumor tissue.

 

Stain Result with Alcian Blue Solution (pH 2.5)

45597.png 

38505.png

Human Large Intestine

 

Double-Staining with Alcian Blue Solution (pH 2.5) and Cold Schiff’s Reagent

45630.png 

*1: Thoroughly wash with distilled water after washing with running water.

*2: Refer to the Schiff’s Reagent page of the PAS-staining protocol (See the page #15).

Staining Result

· Acidic mucopolysaccharides: blue

· Neutral mucus

· Sialomucin, sulfomucin: Reddish purple ~ blue

· Nucleus: bluish purple ~ purplish indigo

 

<Note>

If you prepare the pH of Alcian blue to 1.0, only sulfomucin and acid mucin will stain reddish purple to blue, and neutral mucin will stain bright red.

 

38644.png

Human Stomach

Product Name

Grade

Package Size

Wako Cat. No.

Alcian Blue Solution, pH 2.5

for Pathology Research

500 mL

015-13805

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Toluidine Blue Staining

Acidic mucopolysaccharides are present in the connective tissue (hyaluronic acid and chondroitin sulfate B), cartilage tissue (mucoitin sulfate, chondroitin sulfate A,C), mast cells (heparin), and epithelial mucus, etc.

The staining method is based on the fact that these compounds stain metachromatically with the basic tar dye toluidine blue.

Staining Result

Acidic mucopolysaccharides, mast cell granule:

reddish purple

Nuclei: blue

Sulfur-containing poysaccharides such as chondroitin sulfate show positive metachromasy (reddish purple) at all pH. On the other hand, hyaluronic acid shows a negative metachromasy (blue) at pH 2.5.

Composition

0.05% Toluidine Blue Solution: pH 2.5, 4.1 and 7.0

Reagent 1: 0.1 mol/L Citric Acid Solution

Reagent 2: 0.2 mol/L Disodium Hydrogen Phosphate Solution

 

· Mix Reagent 1 and Reagent 2 in the ratios described below and prepare buffers. Add toluidine blue at a proportion of 0.05% to each solution and dissolve.

pH 2.5 Solution: <Reagent 1> : <Reagent 2> = 19.0: 1.0 (mL)

pH 4.1 Solution: <Reagent 1> : <Reagent 2> = 12.0: 8.0 (mL)

pH 7.0 Solution: <Reagent 1> : <Reagent 2> = 3.5: 16.5 (mL)

Mucus

pH

Acidic Mucopolysaccharide

Hyaluronic Acid

Mucoitin Sulfate

Chondroitin Sulfate

2.5

-

+

4.1

+

+

7.0

+

+

 

Staining Protocol

45663.png 

38779.png

[Fig. 1, 2]

Metachromasy of the umbilical cord

(0.05% Toluidine Blue, pH 4.1)

Metachromasy of hyaluronic acid molecules that have not conjugated with sulfate groups and of acid mucopolysaccharides

[Fig.1] Toluidine Blue, pH 4.1 (×4)

[Fig.2] Toluidine Blue, pH 4.1 (×4)

38785.png

[Fig. 3, 4]

Metachromasy of the tracheal cartilage

(0.05% Toluidine Blue, pH 2.5, pH 4.1)

Enhanced metachromasy of sulfomucopolysaccharides

[Fig.3] Toluidine Blue, pH 2.5 (×4)

[Fig.4] Toluidine Blue, pH 4.1 (×4)

38792.png

[Fig. 5, 6]

Metachromasy of heparin in mast cells

(0.05% Toluidine Blue, pH 4.1)

 

Data was provided by Teiji Takezaki & Masahiko Fujiwara, Pathological and Microbiology Lab, Kotobiken Medical Laboratories

[Fig.5] Toluidine Blue, pH 4.1 (×20)

[Fig.6] Toluidine Blue, pH 4.1 (×40)

Product Name

Grade

Package Size

Wako Cat. No.

0.05% Toluidine Blue Solution (pH2.5)

for Pathology Research

500 mL

202-14535

0.05% Toluidine Blue Solution (pH4.1)

for Pathology Research

500 mL

209-14545

0.05% Toluidine Blue Solution (pH7.0)

for Pathology Research

500 mL

206-14555

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Colloidal Iron Staining

The colloidal iron staining is a method in which colloidal trivalent iron (Fe2O3) is adhered to acid mucopolysaccharides and detected by Prussian blue reaction.

Staining Result

· Elastic fibers, HBs antigens, cartilage matrix: Blue

· Nuclei: Red

· Background: Light pink

 

45689.png 

*1: Colloidal Iron Solution

Distilled Water ……18 mL

Glacial Acetic Acid ……12 mL

Colloidal Iron Stock Solution ……10 mL

Product Name

Grade

Package Size

Wako Cat. No.

Colloidal Iron Stock Solution

for Pathology Research

100 mL

037-11601

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G-3.

Connective Tissue Staining

Elastic Van Gieson’s Stain

Collagen fibers undergo the most rapid growth when damaged connective tissue is being repaired. The standard Van Gieson’s stain is a widely used method of visualizing such collagen fibers. However, most investigators want to stain more than one target. In such cases, the advanced Elastic Van Gieson’s stain is more advantageous and popular because it allows simultaneous histology of elastic, collagen and muscle fibers.

Staining Result

· Elastic fibers: Blackish purple

· Collagen fiber: Red

· Muscle fiber, Erythrocyte, Cytoplasm: Yellow

· Nucleus: Blackish purple

 

45721.png 

Weigert’s Iron Hematoxylin Staining Set

Kit Components

Weigert’s Iron Hematoxylin Solution I ……500 mL × 1 bottle (1% Hematoxylin-96% Ethanol)

Weigert’s Iron Hematoxylin Solution II ……500 mL × 1 bottle (2% Iron (III) Chloride, Hexahydrate- abt. 0.25% Hydrochloric Acid)

Preparation of Reagent

Staining Solution (Prepare just before use)

Mix Solution I and II at a ratio of 1:1.

 

39086.png

Human Kidney

Product Name

Grade

Package Size

Wako Cat. No.

Weigert's Iron Hematoxylin Staining Set

for Pathology Research

1 set

298-21741

Van Gieson Solution P (Picric Acid Solution)

Composition

A solution with picric acid dissolved in distilled water to the saturation point.

 

Van Gieson Solution F (1% Acid Fuchsin Solution)

Composition

Acid Fuchsin ……5 g

Distilled Water ……500 mL

Prepare Van Gieson’s solution just before use. (Van Gieson Solution P : Van Gieson Solution F = 100 mL : 15 mL)

Product Name

Grade

Package Size

Wako Cat. No.

Van Gieson Solution P

for Pathology Research

500 mL

224-01405

Van Gieson Solution F

for Pathology Research

500 mL

221-01415

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Masson’s Ttrichrome Stain

Masson’s trichrome stain is a method of staining to distinguish collagen fibers. Nucleus is stained black with iron hematoxylin, cytoplasm is stained red with Xylidine Ponceau acid fuchsin azofloxin, and collagen fiber is stained blue with aniline blue. It is called “trichrome stain”.

45747.png 

*1:Prolonged treatment will reduce the quality of the aniline blue stain to some extent. The proper treatment time will depend on the thickness of the section.

*2:We recommend staining for a longer duration and differentiating with 0.5 to 1% acid alcohol. If possible, prepare agents I and II when performing the protocol.

Double strength Carazzi’s hematoxylin can be used as a substitute but will require more time for staining compared to iron hematoxylin.

*3:The more effective the 1st mordant solution, the more red the iron hematoxylin will turn out. The 2nd mordant is used to maintain the dark hue, but prolonged treatment will easily diminish the quality of the aniline blue stain. Hence, do not treat the specimen with the 2nd mordant solution for more than one minute.

*4:You may skip the Orange G in this step and replace aniline blue with a mixture of aniline blue and Orange G for AZAN staining. Staining performance will remain virtually the same. Use undiluted aniline blue and Orange G solutions if you decide to employ this method. (0.8% Orange G is also acceptable.)

*5:Staining time for aniline blue will vary depending on the organ, condition and section thickness, so monitor the specimen carefully with microscopy. Treat the specimen with aniline blue again if staining performance is insufficient.

*6:Prolonged treatment to 1% acetic acid hydrate will reduce aniline blue quality. If this happens, prepare two tanks: wash the section in one tank and fix in the other. This will improve the balance between red and blue colorization.

*7:Differentiate each section carefully. Use the AZAN staining protocol as a reference. Isopropyl alcohol is appropriate for differentiating. If you believe you have stained for too long, use pure ethanol which has the effect of somewhat weakening the blue colorization.

Staining Results

· Nuclei: purplish black ~ purplish red

· Collagen Fiber, Glomerular Basement Membrane, Reticular Fiber: bright blue

· Mucus: blue

· Fibrin: red ~ reddish orange

· Basophils: blue

· Eosinophils: red

· Cytoplasm: pale red ~ purplish red

· Red Blood Cell: orangish yellow ~ orangish red

Product Name

Grade

Package Size

Wako Cat. No.

Aniline Blue Solution

for Pathology Research

500 mL

015-18045

0.8% Orange G Solution

for Pathology Research

500 mL

159-02245

Ponceau Xylidine, Fuchsin Acid, Azophloxine Solution

for Pathology Research

500 mL

166-19765

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Azan Stain

Azan stain is a method of staining collagen fibers selectively with aniline blue. At the same time, pathologic products such as hyaline degeneration and fibrin can be stained in different colors besides reticular fibers.

45762.png 

*1:This step is unnecessary if you are using Helly's or Zenker's fixative. Prolonged exposure will reduce the quality of the aniline blue stain.

*2:Stain thoroughly at room temperature. You can raise the temperature to about 60°C if you are in a hurry, but this will degrade the quality of the staining solution.

*3, 4:Differentiate each section individually because exposure to aniline/alcohol leads to faster decolorizing. Differentiate until you can distinguish the cytoplasm and the nuclei, and when the fibers become light red. Staining should be somewhat strong since decolorization will progress with exposure to tungsten phosphate in the next step. If you have stained properly you will not be able to differentiate individual component. Then you should place the section in tungsten phosphate for an extended length of time. This is a key to achieving a color that is not too dark and not too light, and will greatly affect the quality of the aniline stain. Instead of differentiating once, differentiate several times while making sure you stop the reaction with acetic alcohol in between rounds and you monitor progress with microscopy after washings.

*5:This is the mordant solution for aniline blue. Because new solution will result in strong decolorization, use an equal concentration mixture of new and old solution.

*6:Instead of using a mixture, perform Orange G staining and aniline blue staining independently. After mordanting, stain the section in a solution made by adding one drop of glacial acetic acid to 50 ml of 0.75% Orange G. Wash the section lightly with tap water, pass through distilled water and then transfer to azocarmine G-drifting solution. Do not add aniline blue to the Orange G solution. Compared to staining with a mixture, you should notice a prominent Orange G hue. The red blood cells should be yellowish and nuclei and cytoplasm should be brightly colored.

*7:Differentiation with aniline blue will determine whether the AZAN stain is acceptable. Be sure to replace the staining solution with ethanol immediately. Coloring will become dull if the section is passed through water. Wipe away solution from the back side of the slide glass and the surrounding area of the section. Tilt the slide glass and wash away staining solution using a pipette filled with isopropyl alcohol or pure ethanol. Lay the slide glass flat and apply alcohol. Shake the slide very gently and then decant. Repeat this alcohol rinse of the section until you are able to differentiate blue, red and intermediate color stains. You may perform this process in a staining dish if you have many sections, but be sure to replace the alcohol meticulously. If you want to repeat staining, decolorize using a low-concentration ammonia hydroxide solution.

Staining Result

· Nuclei: Dark red

· Fibrin: Red

· Basophils: Blue

· Eosinophils: Red

· Collagen, glomerular basement membrane, reticular fibers, hyaline material, mucus: Bright blue

 

Aniline Blue · Orange G Solution

Composition

Aniline Blue ……0.5 g

Orange G ……2 g

Distilled Water ……100 mL

Acetic Acid ……8 mL

Product Name

Grade

Package Size

Wako Cat. No.

Aniline Blue · Orange G Solution

for Pathology Research

500 mL

012-18055

 

Azocarmine G Solution

Composition

Azocarmine G ……0.1 g

Distilled Water ……100 mL

Acetic Acid ……1 mL

Product Name

Grade

Package Size

Wako Cat. No.

Azocarmine G Solution

for Pathology Research

500 mL

019-18065

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Victoria Blue Staining

Elastic fibers and HBs antigens stain blue. Oxidation and reduction is necessary for pretreatment when staining HBs antigens.

 

Staining Result

· Elastic Fibers, HBs antigens, Cartilage matrix: blue

· Nuclei: Red

· Background: pale pink

45781.png 

Product Name

Grade

Package Size

Wako Cat. No.

Victoria Blue Solution

for Pathology Research

500 mL

223-01475

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G-4.

Staining of Fibrins and Glial Tissues

Phosphotungstic Acid Hematoxylin Staining

The product can stain neuroglia fiber and collagen fiber in different colors, and is also used for staining muscle fiber and fibrin.

It is useful for discriminating neuroglia fiber and connective fiber in central inflammatory disease and brain tumor and for observing the extent of metastasis.

Composition

Hematoxylin ……1 g

Phosphotungstic Acid ……20 g

Distilled Water ……1,000 mL

 

41145.png

45794.png 

Product Name

Grade

Package Size

Wako Cat. No.

Phosphotungstic Acid Hematoxylin Solution

for Pathology Research

100 mL

167-15611

500 mL

169-15615

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G-5.

Intracellular Inorganic Substance Staining

Berlin Blue Staining

This staining is used for staining trivalent iron ion in tissues, especially as hemosiderin staining. Hemosiderin is a yellowish- or brownish-red dye originated from hemoglobin, and found in the tissues and cells after bleeding.

Kit Components

2% Hydrochloric Acid ……500 mL × 1 bottle

2% Potassium Ferrocyanide ……500 mL × 1 bottle

Preparation of Staining Solution:Mix equal amount of each solution just before use.

 

45865.png

Human Spleen

 

45950.png 

Product Name

Grade

Package Size

Wako Cat. No.

Berlin Blue Staining Set

for Pathology Research

1 set

296-21541

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G-6.

Cytological Staining

Papanicolaou Staining

Papanicolaou stain is for cytological diagnosis and is an essential staining method for the early detection and definitive diagnosis of cancer.

Principle of Papanicolaou stain

Nucleus Staining

Hematoxylin stains nuclei. Hematoxylin is converted by an oxidizing agent to hematein, which upon addition of alum will form a chelate bond with aluminum, resulting in aluminum rack. This is positively charged and has staining ability, and upon binding to the negatively charged phosphate group of a nucleic acid will turn it dark purple.

 

46004.png

Adenocarcinoma cells of Sputum

46059.png 

Papanicolaou-Gill’s Hematoxylin (Gill-5) Stain Solution

Composition

Hematoxylin ……5 g

Distilled water ……730 mL

Aluminum sulfate ……44 g

Sodium Iodate ……0.52 g

Ethylene glycol ……250 mL

Product Name

Grade

Package Size

Wako Cat. No.

Papanicolaou-Gill's Hematoxylin (Gill-5) Stain Solution

for Pathologic Cytodiagnosis Research

500 mL

167-19795

 

Other Papanicolaou Staining Solution

Papanicolaou EA100, OG100 and Papanicolaou-Hematoxylin Stain Solution are cytoplasmic nuclear stain solutions of Papanicolaou’s stain commonly used in cytological diagnosis. These have almost the same stain pattern as the conventional one but have been improved so as to retain stain accuracy for a long time. These were prepared according to a protocol developed by Tenjin, et al.

46076.png 

Product Name

Grade

Package Size

Wako Cat. No.

Papanicolaou, Hematoxylin Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

168-18941

Papanicolaou EA100 Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

164-18921

Papanicolaou OG100 Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

161-18931

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G-7.

Blood Cell Staining

Staining Results

Nuclei: Red

Cytoplasm: Blue ~ pale blue

Acidophilic granule: Red

Basophilic granule: purplish blue ~ blue

Erythrocyte: pinkish red

 

Giemsa Stain Solution

Staining Protocol

Fix: Add a few drops of methanol ……abt. 30 sec.

Preparation of Giemsa Staining Solution: Add Giemsa Staining Solution at a ratio of 1 to 1.5 drops per 1 mL of water. (2 ~ 3 mL of the prepared Giemsa Staining Solution will be required for each specimen.)

<Note>Dilute the Giemsa Staining Solution just before use. This is because if the solution is left standing, azure B and eosin Y molecules will bond, resulting in a loss of staining performance.

Staining: Apply the prepared staining solution to the painted side ……15 ~ 30 sec.

Wash with water ……15 ~ 30 sec.

Dry

 

May-Grünwald Stain Solution

Staining Protocol (May-Grünwald, Giemsa Stain)

Fix & Stain: Add 10 drops of May-Gruenwald Stain Solution ……2 ~ 3 min.

Add 10 drops of phosphate buffer ……2 ~ 3 min.

Wash with prepared Giemsa Staining Solution ……2 ~ 3 min.

Wash: prepared Giemsa Stain Solution ……10 ~ 15 min.

Wash with running water

Dry

 

Wright Stain Solution

Staining Protocol 1 (Wright Stain)

Fix, Stain: Add 10 ~ 15 drops of Wright Stain Solution ……2 ~ 3 min.

Add 10 ~ 15 drops of phosphate buffer onto the 1. and mix ……4 ~ 7 min.

Wash with running water ……15 ~ 30 sec.

Dry

 

Staining Protocol 2 (Wright-Giemsa Stain)

Fix & Stain: Add 10 ~ 15 drops of Wright Stain Solution ……2 ~ 3 min.

Add 10 drops of phosphate buffer ……2 ~ 3 min.

Wash: Pour the prepared Giemsa Staining Solution from above ……15 ~ 30 sec.

Stain: prepared Giemsa Staining Solution ……10 ~ 15 min.

Wash with running water

Dry

<Note>Wright’s one step stain provides brightly colored granules, but also drably stained nuclei.

Product Name

Grade

Package Size

Wako Cat. No.

Giemsa Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

079-04391

May-Grünwald Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

131-12811

Wright Stain Solution

for Pathologic Cytodiagnosis Research

250 mL

238-01541

May-Grünwald’s Eosin Methylene Blue Solution (MGE solution)

The solution is used for staining of blood cell components such as erythrocytes, basophil granules, and neutrophil granules in blood and pathological tissues.

Usage

Add this solution into methanol, dissolve it by warming and mixing for 1 hour at 40°C. After slowly-cooling, filter it.

Staining Results

Nuclei: reddish purple

Acidophilic granule: red or reddish brown

Neutrophil granule: reddish purple

Basophilic granule, platelet: Blue

Erythrocyte: pale red

 

41638.png

Leukemic lymph node

* 1:Dilute with distilled water

* 2:Prepare the solution with distilled water (200 mL) + acetic acid (3 drops)

 

46126.png 

Product Name

Grade

Package Size

Wako Cat. No.

May-Grünwald's Eosin Methylene Blue Solution

for Pathology Research

500 mL

138-11645

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G-8.

Hard Tissue Staining

TRAP/ALP Stain Kit

Normal bone metabolism is based on the balance between bone formation by osteoblasts and bone resorption by osteoclasts. When the balance is disturbed and bone resorption by osteoclasts is abnormally increased, bone mass is reduced leading to osteoporosis.

Therefore, various researches have been carried out to understand the mechanism of osteoclast and osteoblast metabolism, and to utilize this knowledge for the treatment of the diseases and for the development of effective drugs.

Today, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) are known as marker enzymes for osteoblasts and osteoclasts, respectively, and these enzymes are used as one of the markers showing the presence of osteoblasts and osteoclasts in tissue sections or cultured cells.

This kit enables you to examine the state of differentiation of bone cells and the cell distribution in bone tissues by observation of the stained images of osteoblasts and osteoclasts in the tissues and cultured cells using ALP/TRAP enzyme activities in the tissue sections and cultured cells.

By studying the stained image of osteoblasts and osteoclasts, you can track their distributions and differentiation status in bone tissue.

 

Features

 

41682.png 

*Osteoclasts stain red with TRAP staining solution

*Chondrocytes, intercellular membranes and the cell membranes of osteoblasts stain brownish-red with ALP staining solution.

*Nuclei of various cells stain bluish green with nuclear staining solution.

Kit Components

Sodium tartrate soln. (×10) ……3 mL × 1 bottle

TRAP substrate soln. A ……30 mL × 1 bottle

TRAP substrate soln. N ……0.3 mL × 1 bottle

Nuclear stain soln. ……10 mL × 1 bottle

ALP substrate soln. (premixed) ……30 mL × 1 bottle

<Note>This kit contains enough reagent to stain an equivalent of five 24-well culture plates, six 96-well culture plates, or 60 bone tissue slides (when using 500 μL perslide).

 

Activity Staining of TRAP/ALP in Cultured Cells

41721.png 41727.png

 

Activity Staining of TRAP

in RAW 264 cells

Activity Staining of ALP

in MC 3T3-E1 cells

Product Name

Grade

Package Size

Wako Cat. No.

TRAP/ALP Stain Kit

for Pathology Research

60 tests

294-67001

PAGE TOP

G-9.

Other Stains

Stain Product List

Product Name

Grade

Package Size

Wako Cat. No.

Acridine Orange (CI 46005)

Practical Grade

5 g 014-08941
25 g 012-08942

Acridine Yellow

-

25 g

018-00742

Aniline Blue · Water Soluble [Water Blue]

-

25 g

016-21302

Auramine (CI 41000)

Wako 1st Grade

10 g

013-04871

Bismarck Brown (CI 21000)

Wako 1st Grade

25 g 028-01902

Carmine (CI 75470)

Wako Special Grade

5 g

035-01371

25 g

033-01372

Chlorazol Black E

for Protozoan Kohn's Staining

25 g 038-17432

Congo Red (CI 22120)

JIS Special Grade

25 g

032-03922

Crystal Violet (CI 42555)

JIS Special Grade

25 g 031-04852

for Pathology Research

25 g 038-17792

4',6-Diamidino-2-phenylindole Dihydrochloride n-Hydrate [DAPI]

for Biochemistry

5 mg

043-18804

10 mg

049-18801

100 mg

045-18803

Eosin Y (CI 45380)

Wako Special Grade

25 g 058-00062

for Pathology Research

25 g 056-06722

Erythrosine (CI 45430)

for Microscopy

25 g

053-00252

Fast Blue RR Salt (CI 37155)

-

5 g 061-03711

25 g 069-03712

Fuchsin Acid (CI 42685)

Practical Grade

25 g

061-01332

Fuchsine Basic (CI 42510)

Wako Special Grade

10 g 066-00581

25 g 064-00582

Gentian Violet R [Methyl Violet]

Wako Special Grade

25 g

136-03272

Hematoxylin Monohydrate (CI 75290)

for Cell Staining

5 g 088-07461
25 g 086-07462

Indigo Carmine (CI 73015)

for Pathology Research

25 g

093-04732

Light Green SF Yellowish (CI 42095)

-

25 g 124-04632

Malachite Green Oxalate

for Pathology Research

25 g

131-13732

Metanil Yellow

Wako Special Grade

25 g 137-01502

Methyl Green

-

10 g

134-13901

25 g

132-13902

Methyl Violet B (CI 42535)

Wako Special Grade

25 g 130-03292

Methylene Blue Tetrahydrate (CI 52015)

for Vital Staining

25 g

137-06982

500 g

131-06985

Neutral Red (CI 50040)

Wako Special Grade

25 g 140-00932

Nile Blue Hydrogensulfate

for Pathology Research

25 g

141-06822

Nile Red

for Pathology Research

25 g 144-08811
100 mg 140-08813

Oil Red O

for Pathology Research

25 g

154-02072

Orange G (CI 16230)

Wako 1st Grade

25 g 150-01832

for Pathology Research

25 g 152-02252

Orcein

Practical Grade

1 g

157-00943

5 g

151-00941

Phloxine B (CI 45410)

Wako Special Grade

25 g 166-02072

Pyronine Y (CI 45005)

Practical Grade

5 g

164-11581

25 g

162-11582

Safranine (CI 50240)

Wako Special Grade

25 g 196-00032

Sirius Red (CI 35780)

for Pathology Research

10 g

196-16201

25 g

194-16202

Sudan 1 (CI 12055)

Wako 1st Grade

25 g 195-04382

Sudan 2 (CI 12140)

Wako Special Grade

25 g

198-06072

Sudan 3 (CI 26100)

Wako Special Grade

25 g 192-04392

Sudan 4 (CI 26105)

Wako 1st Grade

25 g

194-07652

Sudan Black B (CI 26150)

Wako 1st Grade

25 g 192-04412

Thioflavin T (CI 49005)

-

25 g

202-01002

Thionin Acetate

for Pathology Research

5 g 208-18611
25 g 206-18612

Victoria Blue B (CI 44045)

Wako 1st Grade

25 g

228-00222

Victoria Blue 4R (CI 42563)

Wako 1st Grade

25 g 223-00772

Wright Stain, Powder

Wako Special Grade

10 g

235-00191

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H.

Mounting Reagent

H-1.

Softmount

A xylene-free oil-soluble mounting Reagent

Softmount is a very safe high-performing mounting reagent that does not contain hazardous xylene.

Adjust Softmount viscosity by diluting with Lemosol® A.

Features

Product Name

Grade

Package Size

Wako Cat. No.

Softmount

for Pathology Research

250 mL

192-16301

<Related Products>

Product Name

Grade

Package Size

Wako Cat. No.

Apathy's Mounting Media, Soluble

for Pathology Research

100 mL

010-13811

Canada Balsam

Wako 1st Grade

25 g

034-01042

100 g

036-01041

500 g

038-01045

PAGE TOP

I.

Antigen Retrieval Reagent

I-1.

ImmunoSaver

ImmunoSaver is an antigen retrieval reagent for formalin-fixed tissues. Formalin-fixed tissues are known to form a methylene bridge between proteins, masking the antigenic site and reducing antigenicity. The antigenicity can be recovered by using this product. Conventional methods required consideration in a variety of conditions. However, this product can activate a wide range of antigens under the same conditions.

Features

Formulation

10% Citraconic Acid Sodium Solution

Procedure

Dilute ImmunoSaver 200 times with distilled water or deionized water, and then heat to 98°C.

Place the deparaffinized tissue sections into the solution and incubate for 4 5 minutes at 98°C.

Wash 3 times in PBS for 5 minutes and then perform immunostaining.

Data

42202.png

Human Pancreas SNAP-25

1)Treat formalin-fixed paraffinized sections of the human pancreas with ImmunoSaver.

2)Wash once with RBST (PBS containing 0.2% Triton X-100), and twice with PBS.

3)Perform blocking at room temperature for one hour.

4)Rinse lightly with PBS and react with PBST-diluted primary antibody (rabbit anti-SNAP-25 at 1:8,000) at 4°C for one night.

5)Wash once with PBST and twice with PBS.

6)React with biotinylated secondary antibody for one hour at room temperature.

7)Wash once with PBST and twice with PBS.

8)React with peroxidase-labeled avidin (diluted with PBS at 1:400) for one hour at room temperature.

9)Wash once with PBST and twice with PBS.

10)Produce color in the matrix with DAB

11)Wash three times with distilled water, using five minutes per wash.

12)Stain with Mayer’s acid hemalum for one to three minutes.

13)After washing in running water, rinse two to three times with distilled water and then differentiate with 70% ethanol.

14)Clear and mount.

 

Product Name

Grade

Package Size

Wako Cat. No.

ImmunoSaver

for Immunohistologic Staining

25 mL

097-06192

 

J.

Gene-Related Kits

J-1.

Apoptosis in situ Detection Kit Wako

Apoptosis Detection Kit Using the TUNEL Method

This kit is for detection of apoptotic cells. It uses the TUNEL method for detection. The kit contains main reagents ready for use and is suitable for quick and easy analysis. It can be used with paraffin-embedded sections, cultured cells (neutral buffered formalin fixed), and cryosections.PAGE TOP

 

Features

 

Kit Contents

Protein Digestion Enzyme ……1 mL × 1 vial

TdT ……40 μL × 1 vial

TdT Substrate Solution ……4.4 mL × 1 vial

100 × POD-Conjugated Antibody ……44 μL × 1 vial

DNase I ……4 μL × 1 vial

10 × DNase I Reaction Buffer ……40 μL × 1 vial

<Note>The kit does not contain a chromogenic substrate. We recommend GenWay’s two-component liquid substrate, DAB Immunohistochemistry Substrate (#45-053-150038).

 

Application

Transplanted breast cancer in a mouse treated with a-Mangostin (× 200)

42246.png

Control group

a-Mangostin group

Apoptosis of carcinoma cells due to antitumor effect by a-Mangostin was confirmed .

(Data was provided by Professor Masaaki Shibata, Osaka Health Science University. )

 

Product Name

Grade

Package Size

Wako Cat. No.

Apoptosis in situ Detection Kit Wako

for Apoptosis Research

40 tests

293-71501

<Related Products>

Product Name

Grade

Package Size

Wako Cat. No.

Apoptosis Ladder Detection Kit Wako

for Apoptosis Research

24 lanes

297-71401

96 lanes

293-71403

DAB Tablet (DAB. 4HCl 10 mg/Tablet)

for Biochemistry

50 tablets

049-22831

100 tablets

045-22833

DAB Tablet (DAB. 4HCl 5 mg/Tablet)

for Biochemistry

50 tablets

040-27001

100 tablets

046-27003

DAB TRIS Tablet, pH 7.6

for Biochemistry

50 tablets

047-27011

PAGE TOP

J-2.

DNA Isolator PS Kit

DNA Isolation from Paraffin-embedded tissue sections

This kit can isolate DNA from paraffinized sections in a short length of time.

 

Features

Kit Contents

Enzyme reaction solution ……2 mL × 1 vial

Enzyme Activator ……34 mg × 1 vial

Protease ……2.2 mg × 1 vial

Accelerator for DNA Precipitation ……0.5 mL × 1 vial

Dilution Solution for DNA Amplification ……0.5 mL × 1 vial

(Deparaffinizing reagent such as Lemosol® A (Wako Catalog No. 120-04411) is necessary additionally. (See page #9))

Data

46216.png 

Product Name

Grade

Package Size

Wako Cat. No.

DNA Isolator PS Kit

for Genetic Research

100 tests

295-52401

PAGE TOP

 

J-3.

DNA Isolator PS-Rapid Reagent

DNA extraction from paraffin-embedded sections in 20 minutes.

Features

Kit Components

DNA Isolation Solution ……10 mL × 5 drop-style tubes

(One drop from the eye drop-style tube contains about 30 μL of solution)

Data

Amplification of the b-globin gene using DNA isolated from paraffinized sections of various tissue specimens as templates.

 

42503.png

M: f X174/Hae III 1: b-globin (205 bp) 2: b-globin (325 bp) 3: b-globin (408 bp)

Product Name

Grade

Package Size

Wako Cat. No.

DNA Isolator PS-Rapid Reagent

for Genetic Research

100 tests

291-56401

<Related Products>

Product Name

Grade

Package Size

Wako Cat. No.

Ethanol (99.5)

for Molecular Biology

100 mL

052-07221

500 mL

054-07225

2-Propanol

for Molecular Biology

100 mL

166-21671

500 mL

168-21675

PAGE TOP

 

J-4.

ISOGEN PB Kit

RNA Extraction Kit from Paraffin-Embedded Tissue Sections

The ISOGEN PB Kit is for extracting RNA from paraffin-embedded tissue sections.

RNA can be extracted in 2 hours by the following simple manipulations:

Deparaffinization Proteinase K treatment RNA extraction

Use of Extracted RNA

Kit Contents (for 20 tests)

Proteinase K (20 mg/mL)……100 μL × 1

Extraction Buffer……3 mL × 1

ISOGEN-LS……10 mL × 1

Ethachinmate……60 μL × 1

Deoxyribonuclease (RT Grade)……20 μL × 1

10 × DNase (RT Grade) Buffer II……100 μL × 1

Stop Solution (RT Grade)……100 μL × 1

DEPC treated Water……500 μL × 2

Reagents necessary in addition to the kit

Lemosol® or Lemosol® A, Ethanol, Chloroform, Isopropanol

42604.png

RNA Extraction Protocol

42615.png 

42622.png 

Lane 1: OneSTEP Marker 5 (φX174/Hinc II digest)

Lane 2: No template

Lane 3: Template = RNA without reverse transcription reaction

Lane 4: Template = RNA with reverse transcription reaction

Lane 5: OneSTEP Marker 5 (φX174/Hinc II digest)

 

Application

Detection of Gapd gene by RT-PCR

RNA was extracted using 10 μm × 4 sheets of paraffin-embedded tissue section from mouse submaxillary gland in accordance with the protocol. The 500 ng of obtained RNA was treated with DNase by the protocol, followed by detection of mouse Gapd gene exon 5 (258 bp) by RT-PCR.

Product Name

Package Size

Wako Cat. No.

ISOGEN PB Kit <manufactured by Nippon Gene Co., Ltd>

20 tests

315-06421

<Related Products>

Product Name

Package Size

Wako Cat. No.

ISOGEN-LS <manufactured by Nippon Gene Co., Ltd>

10 mL

317-02623

100 mL

311-02621

PAGE TOP

K.

Paraffin-embedded Tissue Sections

K-1.

HistoMap Series

HistMap series are paraffin-embedded tissue sections from normal rat or mouse fixed for 3 days in 10% neutral buffered formalin and can be used for HE staining, immunostaining and other types of staining protocols.

 

HE Staining

46399.png 

Section photo

43007.png

Please remove the protective seal on the product before use.

Paraffin-embedded Tissue Section from mouse

· ICR mouse (SPF animal)
· Age, Sex: 10 week-old, male
· Thickness: abt. 3 μm
Fixing: 10% neutral buffered formalin

Product Name

Grade

Package Size

Wako Cat. No.

HistoMap Mouse Normal Cerebellum, Paraffin Embedded Tissue

for Pathology Research

10 sheets

089-09571

HistoMap Mouse Normal Cerebrum, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

082-09561

HistoMap Mouse Normal Heart, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

087-09491

HistoMap Mouse Normal Kidney, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

081-09531

HistoMap Mouse Normal Liver, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

086-09581

HistoMap Mouse Normal Lung, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

087-09511

HistoMap Mouse Normal Pancreas, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

084-09521

HistoMap Mouse Normal Rectum, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

080-09481

HistoMap Mouse Normal Spleen, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

080-09501

HistoMap Mouse Normal Stomach, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

088-09541

HistoMap Mouse Normal Testis, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

085-09551

 

Paraffin-embedded Tissue Section from rat

· F344/DuCr1j (SPF animal)
Age, Sex: 10 week-old, male
· Thickness: abt. 3 μm
Fixing: 10% neutral buffered formalin

Product Name

Grade

Package Size

Wako Cat. No.

HistoMap Rat Normal Cerebellum, Paraffin Embedded Tissue

for Pathology Research

10 sheets

080-09621

HistoMap Rat Normal Cerebrum, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

081-09651

HistoMap Rat Normal Heart, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

087-09631

HistoMap Rat Normal Kidney, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

084-09641

HistoMap Rat Normal Liver, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

083-09611

HistoMap Rat Normal Lung, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

085-09671

HistoMap Rat Normal Pancreas, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

089-09691

HistoMap Rat Normal Rectum, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

088-09661

HistoMap Rat Normal Spleen, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

082-09681

HistoMap Rat Normal Stomach, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

086-09601

HistoMap Rat Normal Testis, Paraffin Embedded Tissue Section

for Pathology Research

10 sheets

082-09701

 

 

46468.png