Pam,

Cytochrome oxidase enzyme histochemistry is used to detect defects of
cytochrome oxidase activity, and to demonstrate mitochondria in skeletal
muscle bxs. Cytochrome oxidase is frimly bound to mitchondrial membranes
and to some extent, the enzyme activity is an indication of the number
of mitochondria and oxidative metabolism within the cells.
Type I fibers have higher oxidative metabolism than type II.

Methodology:

Snap-frozen, unfixed cryostat sections, 10 micron	

 

Incub Medium:
3,3'-diaminobenzidine tetrahydrochloride	20mg (available in tab form
from Sigma)
0.1M phosphate buffer pH 7.4			20ml
Catalase solution				 2ml
Cytochrome C (type II)	- Sigma			20mg

Catalase solution:
Catalase					 4mg
distilled water					100ml

Method:
1. Incubate sections at 37 C			60-120 minutes
2. Rinse in distilled water
3. Fix in formol calcium			5-10 minutes
4. Dehydrate,clear and mount in synthetic mountant

Results:
Sites of enzyme activity -brown granular reaction product.
Type I activity higher than Type II fibers

Note:
Cytochrome oxidase is very sensitive to fixation.

Reference:
Seligman AM, Karnovsky MJ, Wasserkrug HL, Hunker JS.
Nondroplet ultrastructural demonstration of cytochrome oxidase activity
with a polymerising osmiophilic reagent, diaminobenzidine (DAB).
J Cell Biol 1968; 38: 1.

Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
2222 Welborn Street
Dallas, TX 75219
(214) 559 7744
(214) 559 7768 - fax