Semipermeable membrane techniques in quantitative enzyme histochemistry

Non-structurally bound, partially structurally bound or weakly structurally bound enzymes diffuse out of sections during the histochemical incubation period. Fixation of sections does not help to prevent diffusion since the enzymes may be inactivated by their chemical reaction with the fixative. Moreover watery fixatives cause partial leakage of enzymes into the fixative solution. The inclusion of macromolecular substances in the incubation media only partially prevents the leakage of enzymes. In the technique discussed here a semipermeable membrane is interposed between the incubating solution and the tissue section. Because only molecules with a molecular weight of 20 000 can penetrate the membranes, diffusion of enzymes is prevented, whereas substrate molecules and other components necessary for the staining reaction diffuse through the membrane. This technique is suitable for the histochemical demonstration of activity of oxidoreductases, transferases, hydrolases and isomerases. Particular attention is paid to how far semipermeable membrane methods are suitable for quantitative histochemical studies of enzymes.