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TRADUZIONE di Natale Marzari
 



Colorazione PROTOCOLLO COLORAZIONE TRICROMICA DI GOMORI

(ENGEL-CUNNINGHAM MODIFICATION)

PRINCIPLE:

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

SPECIMEN REQUIRED:

Snap frozen human striated muscoli. (Use the isopentane freezing method precedentemente described.)

METHOD:

Fixation: None, use snap frozen tissue.

Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from snap frozen Biopsia. Attach one o più sections to a No.1½, 22 mm squsono coverslip.

Equipment:
Ceramic Colorazioneing rack - Thomas Scientific #8542-E40
Columbia Colorazioneing dish - Thomas Scientific #8542-C12
Columbia Colorazioneing 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 Colorazione, acidified, - Lerner laboratori * #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 - istochimiche Fisher A962-4, o 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 Colorazione
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 usando 1 N NaOH

Store at room temperature, prepare weekly

2. Acetic acid, ~0.2 %
Deionized water 1000 ml
Acetic acid, glacial 2 ml

3. Alcol 50 %
Reagent alcohol ~50 ml
Deionized water ~50 ml

4. Alcol 70 %
Reagent alcohol ~70 ml
Deionized water ~30 ml

5. Alcol 80 %
Reagent alcohol ~80ml
Deionized water ~20 ml

6. Alcol 95 %
Reagent alcohol ~95 ml
Deionized water ~ 5 ml

Colorazioneing Procedure:

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

  2. Immerse sections in Harris Hematoxylin for 5 minutes.

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

  4. Immerse sections in Colorazione tricromica di Gomori for 10 minutes.

  5. Differenziate usando 0.2% acetic acid. A few dips should be sufficient.

  6. Immerse rack con sections directly into 95 % alcohol

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

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

  9. Mount coverslip onto a labeled glass slide con Permount o some other suitable organic mounting medium.
Results:
  • Nuclei: Red-purple
  • Muscoli normali myofibrils: Green-blue con distinct A e I bands
  • Intermyofibrillar muscoli membranes: Red
  • Interstitial collagen: Green

REFERENCES:

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

2. Sheehan, D.C. e Hrapchak, B.B. THEORY AND PRACTICE OF HISTOTECHNOLOGY, Battelle Memorial Institute, Columbus, OH, 1987.
PREPARED BYG. JAMES PLANER 09/21/90
SUPERCEDES REVISION G. JAMES PLANER 06/30/95
REVISED BYKAREN BIESER 07/14/97
ADOPTED BYALAN PESTRONK, M.D.
REVIEWED BY
REVIEWED BY
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