Neuromuscular 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 m) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No.1, 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:

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