The calcium method for ATPase demonstration, employing solutions of different pH values, have been used primarily to distinguish muscle fiber types. The way this stain is believed to work is as follows. The remaining active enzyme is attached to a calcium atom which is replaced by a cobalt and finally precipitated as a black insoluble compound by the ammonium sulfide. This is a complicated stain and there are several areas in which one needs to be careful in order to achieve a good fiber type differentiation. The pH of all solutions is critical.
Timing is crucial.
As it ages or oxidizes, it becomes more red and cannot be used. Snap frozen human striated muscle. None, use snap frozen tissue. 100 ml Store at room temperature 7. N Hydrochloric Acid 10 ml 1 N HCl to 90 ml deionized water Store at room temperature 10.