Three approaches have been investigated for the histochemical demonstration of acid and alkaline phosphatases with the substrate 2-naphthylthiolphos-phate-bisdicyclohexylamine salt, NTP. Each method results in the formation of osmiophilic end-products which are converted to osmium blacks at the sites of acid or alkaline phosphatase activity. Method A gives an osmium black via an intermediate osmiophilic diazothioether formed by capture of enzymatically liberated 2-naphthalenethiol with the diazonium salt, Fast Blue BBN. The other two methods depend upon capture of 2-naphthalenethiol with either ionic cadmium (Method B) or ionic lead (Method C) and subsequent osmication of the cadmium mercaptide or lead mercaptide.
In light microscopy, both Methods A and B give better results than Method C for acid phosphatase and especially so for alkaline phosphatase. More enzymatic inhibition of non-lysosomal acid phosphatase is noted with Methods B and C. In electron microscopy, Method A yields deposits in droplet form which are too large to relate properly to underlying ultrastructure. Methods B and C yield deposits that are too crystalline to be useful for relation to fine structure.