CLINICAL LABORATORY

GOMORI TRICHROME STAIN PROTOCOL

(ENGEL-CUNNINGHAM MODIFICATION)

PRINCIPLE:

Gomori's one-step trichrome is a staining procedure that combines the plasma stain (chromotrope 2R) and connective fiber stain (fast green FCF) in a phosphotungstic acid solution to which glacial acetic acid has been added.

SPECIMEN REQUIRED:

Snap frozen human striated muscle. (Use the isopentane freezing method previously described.)

METHOD:

Fixation: None, use snap frozen tissue.

Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No.1½, 22 mm square coverslip.

Equipment:

Ceramic staining rack - Thomas Scientific #8542-E40

Columbia staining dish - Thomas Scientific #8542-C12

Columbia staining dish(jar) - Thomas Scientific #8542-E30

Forceps

Latex gloves

Reagents:

Glacial Acetic Acid -Fisher A507-500, CORROSIVE store at room temperature

Chromotrope 2R - Sigma C3143, IRRITANT, store at room temperature

Deionized water

Fast Green FCF - certified, Sigma F7258, GLOVES AND MASK REQUIRED, store at room temperature

Harris Hematoxylin Stain, acidified, - Lerner Laboratories * #1931382, store at room

Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD

Phosphotungstic acid, free acid, - Sigma P4006, CORROSIVE, store at room temperature

Reagent alcohol, ACS - histochemical Fisher A962-4, or HPLC A995 FLAMMABLE, TOXIC, TERATOGENIC, store at room temperature in flammable cabinet

Xylenes - Fisher #HC700-1GAL, FLAMMABLE, store room temperature in flammable cabinet)

Solutions:

I. Gomori's trichrome stain

Chromotrope 2R 0.6 g

Fast green FCF 0.3 g

Phosphotungstic acid 0.6 g

Deionized water 100 ml

Acetic acid, glacial 1.0 ml

Adjust pH of the above mixture to 3.4 using 1 N NaOH

Store at room temperature, prepare weekly

2. Acetic acid, ~0.2 %

Deionized water 1000 ml

Acetic acid, glacial 2 ml

3. Alcohol 50 %

Reagent alcohol ~50 ml

Deionized water ~50 ml

4. Alcohol 70 %

Reagent alcohol ~70 ml

Deionized water ~30 ml

5. Alcohol 80 %

Reagent alcohol ~80ml

Deionized water ~20 ml

6. Alcohol 95 %

Reagent alcohol ~95 ml

Deionized water ~ 5 ml

Staining Procedure:

1. Place the coverslip with section in a ceramic staining rack (Thomas Scientific #8542-E40).

2. Immerse sections in Harris Hematoxylin for 5 minutes.

3. Wash with tap water until the water is clear.

4. Immerse sections in Gomori trichrome stain for 10 minutes.
   

5. Differentiate using 0.2% acetic acid. A few dips should be sufficient.
   

6. Immerse rack with sections directly into 95 % alcohol
   

7. Continue to dehydrate in ascending alcohol solutions (95% x 2, 100% x 2) in columbia staining dish(jar)s - Thomas Scientific #8542-E30.
   

8. Clear with xylene (3 - 4 x ) also in columbia staining dish(jar)s - Thomas Scientific #8542-E30.
   

9. Mount coverslip onto a labeled glass slide with Permount or some other suitable organic mounting medium.
   

Results:

• Nuclei: Red-purple
• Normal muscle myofibrils: Green-blue with distinct A and I bands
• Intermyofibrillar muscle membranes: Red
• Interstitial collagen: Green

REFERENCES:

1. Thompson, Samuel W. SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966.

2. Sheehan, D.C. and Hrapchak, B.B. THEORY AND PRACTICE OF HISTOTECHNOLOGY, Battelle Memorial Institute, Columbus, OH, 1987.

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7/9/2008