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Giunzioni neuromuscolari, Nervi, Spinale, Atassia, Anticorpi e Biopsia, Informazioni per i pazienti
venerdì 21 ottobre 2011
| Natale Marzari | e-mail fonama@fonama.org | Tel. 0039 335 250742 |
|
Giunzioni neuromuscolari, Nervi, Spinale, Atassia, Anticorpi e Biopsia, Informazioni per i pazienti venerdì 21 ottobre 2011 |
|
PRINCIPLE:
citocromo ossidasi is the collective name for part of the oxidative respiratory chain of enzymes localizzato exclusively in the mitocondri of cells. Sometimes cytochrome oxidase was regarded as synonymous con cytochrome a3. This method is a modification of the "Nadi" reaction. The use of 3,3' diaminobenzidine (DAB) results in a brown insoluble compound at the site of cytochrome oxidase activity.
SPECIMEN REQUIRED:
Snap frozen human striated muscoli. (Use the isopentane freezing method described precedentemente.)
Controls: Use myocardium for a positive control. For a negative control, cyanide o sodium azide Inibisce cytochrome oxidase activity.
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.1 1/2, 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:
Catalase (Sigma C-10)
Cytochrome C (Sigma C-2506)
Deionized water
3, 3' diaminobenzidine tetrahydrochloride (Sigma D-5637)
CARCINOGENIC, store
at -20°C
Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD
Reagent alcohol, ACS istochimiche Fisher A962-4,o HPLC A995
FLAMMABLE, TOXIC, store a temperatura ambiente. in flammable cabinet
Sodium dibasic phosphate (Na2HPO4) anhydrous, ACS (FW 141.96)
Sigma S9763 o Fisher S374, o Mallinckrodt 7917;
Store at room temperature
Sodium dibasic phosphate (Na2HPO4)hexptahydrate (FW 268.07)
Sigma S9390,o Fisher S373; Store at room temperature
Sodium monobasic phosphate (NaH2PO4)monohydrate (FW 137.99) ACS
Sigma 9638 o Fisher S369, o Mallinckrodt 7892; Store at room temperature
Sucrose - Sigma S7903, store at room temperature
Xylenes - Fisher #HC700-1GAL, FLAMMABLE, store room temperature in flammable cabinet
Solutions:
I. 0.2 M Fosfato Buffer, pH 7.6
II. Incubating Solution:
III. Alcol 50 % (v/v)
IV. Alcol 70 %(v/v)
V. Alcol 80 % (v/v)
VI. Alcol 95 % (v/v)
Colorazioneing Procedure
1. Place coverslips in the incubating solution in a columbia Colorazioneing
dish (Thomas Scientific #8542-C12) for 60 minutes at room
temperature.
2. Wash con three exchanges of tap o deionized H2O.
3. Dehydrate in ascending alcohols (50%,70%,80%,95% x 2, 100% x 2) in columbia Colorazioneing dish(jar) - Thomas Scientific #8542-E30.
4. Clear con at least 2 changes of xylene columbia Colorazioneing dish(jar) - Thomas Scientific #8542-E30
5. Mount con PERMOUNT o another synthetic organic mounting medium.
Results
Sites of cytochrome oxidase activity sono colored brown.
REFERENCE
1. Seligman et al., 1968. In: HISTOCHEMISTRY - THEORETICAL e APPLIED, A.G. Everson Pearse, Volume. 2, 3a Edition; Williams e Wilkins, Baltimore, 1972.
| PREPARED BY: | G. JAMES PLANER | 10/22/90 |
| SUPERCEDES REVISION | G. JAMES PLANER | 06/20/95 |
| REVIEWED BY | KAREN BIESER | 07/18/97 |
| ADOPTED | ALAN PESTRONK ,M.D. | |
| REVIEWED: | ||
| REVIEWED: |
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