Myofibrillar ATPase Stain

This stain allows a clear discrimination between  three fiber types, one slow-twitch fibre, type I (white fibres) and two fast-twitch fiber types, types IIA (light blue) and IIB (dark blue).


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. Wash twice for 1 min each with Tris-Ca2+ (Pre-Rinse Solution).

3. Incubate for 5 min in the Alkaline Pre-Incubation Solution.

4. Wash twice for 30 sec each with Tris-Ca2+ (Pre-Rinse Solution).

5. Incubate for 90 min at 37 °C in the Incubation Solution.

6. Wash 4 times for 20 – 30 sec each in CaCl2 Wash Solution.

7. Rinse for 3 min in a 2% CoCl2 Solution (mix immediately before use).

8. Rinse 4 times for 20 – 30 sec each in dH2O.

9. Stain for 28 sec in a 1% Azure Stain (mix immediately before use, see notes).

10. Rinse continuously under tap water for 5 min to rinse off remaining Azure Stain.

11. Rinse once with dH2O.

12. Wash with:                  50 % Ethanol for 5 to 10 sec

         70% Ethanol for 5 to 10 sec

         96% Ethanol for 5 to 10 sec

         absolute Ethanol for 1 to 2 min

13. Fix sections using a 1:1 (v:v) solution of Xylol:absolute Ethanol (dispose of in proper receptacle after use).

14. Mount a cover slip using a non-aqueous cover slip medium (i.e. Histofluid).


Solutions and Reagents:

 Pre-Rinse Solution (Fresh Daily) 100 mL 0.18 M CaCl2

(pH 7.3)                                           12.1 g Trishydroxymethylaminomethane

                                                        add dH2O to 1000 mL 

Alkaline Pre-Incubation Solution         10 g CaCl2

(pH 10.4 with 1 N NaOH)                        7.44 g Glycine

                                                                   100 mL Formaldehyd (37%)

                                                                    add dH2O to 1000 mL

 Incubation Solution (Fresh Daily)              20 mL 0.1 M Glycine Buffer

(pH 9.4, warm to 37 °C)                                  10 mL 0.18 M CaCl2 (2%)

                                                                            0.152 g ATP

                                                                            add dH2O to 100 mL

 0.1 M Glycine Buffer                           125 mL Glycine (7.51g/250 mL dH2O)

(pH 9.4)                                                      42 mL 0.4 M NaOH (8 g/250 mL dH2O)

                                                                    add dH2O to 500 mL

 0.18 M CaCl2                            19.98 g CaCl2 in 1000 mL dH2O

 CaCl2 Wash Solution                                    13.3 g CaCl2 in 1000 mL dH2O 


- Azure A is the most  commonly used for this purpose. The 1% Azure A stain is made by  dissolving 1 g of Azure A in 100 mL water.

-  Unless otherwise noted solutions are to be kept at 4 °C, are good for up to one month, and can be disposed of down the sink.



Brooke, M. H., and  K. K.  Kaiser. 1970. Muscle fiber types: how many and what kind ? Arch. Neurol. 23:369-379.

 Szentkuti, L., and A. Eggers. 1985. Eine zuverlässige Modifikation der Myosin-ATPase-Reaktion zur histochemischen Darstellung von drei Fasertypen in der Skelettmuskulatur von Schweinen. Fleischwirtsch. 65:1398-1404.