An improved procedure for the histochemical demonstration of cathepsin D by the mercury-labeled pepstatin method

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.