Mitochondria (Nitro BT) Stain

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). 


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


- Unless otherwise noted solutions are to be kept at 4 °C and can be disposed of down the sink.


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