This stain
preferentially locates muscle fiber mitochondria. Muscle fiber type can thus
be distinguished by the density of mitochondria.
Discrimination is observed between
three fiber types, oxidative fibers, type I (high density),
glycolytic fibers, type IIB (low density), and an intermediate fiber type,
type IIA (medium density).
Procedure:
1. Cut tissue sample on a microtome 10 to 12
mm
thick and place onto microscope slides (microscope slides should be
pre-rinsed/cleaned in an ethanol solution).
Leave slides at room temperature for at least 5 min.
2. Fix sections in 4% formaldehyde-Ca2+ for 45 sec (muscle
samples 24 h post mortem) or 5 min (muscle samples 30 min post mortem).
3. Rinse for 5 min in dH2O.
4. Incubate sections in Staining Medium for 60 min at 37 °C.
5. Rinse for 5 min in dH2O.
6. Mount a cover slip using an aqueous cover slip medium (ie. Glycerine
Gelatine).
Staining Medium
(Fresh Daily)
NADH-Na2
16 mg
Sodium Phosphate Buffer 0.1M (pH 7.4)
3.2 mL
Nitro-BT (4-nitro blue
tetrazolium chloride)
(1 mg/mL) 4.0 mL
DH2O
4.8 mL
Notes:
- Unless otherwise noted solutions are to be kept at 4 °C and can be
disposed of down the sink.
Reference
Novikoff, A; Shin, W.Y.; Drucker, J. (1961) Mitochondrial localization of
oxidative enzymes: Staining results with two tetrazolium salts. Journal of
Biophysical and Biochemical Cytology 9: 47-61