I have been trying to localise alkaline phosphatase on thawed cryosections
of blood neutrophils using the method of John Robinson. The basic reaction
mixutre includes : tricine, TAPS, MgSO4, beta-glycerophosphate, and cerium
chloride [as the capture metal to make the product precipitate and render
it electron dense]. So far, no luck whatsoever. The reaction works fine
in a test tube if I add pure AlkPhosphatase, but not on cryosections. I
have done a dot-blot on nitrocellulose adsorbed alkphosphatase that was
fixed by 2% paraformaldehyde, 0.05% glut, pH 7.2 for varying lengths of
time, up to 1 hour - the activity of pure enzyme is unaffected. Has anyone
else tried this method, or any other histochemical method that was
performed on 50 nm sections for TEM??
Any input would be greatly appreciated.