A PREEMBEDDING TECHNIQUE FOR IMMUNOCYTOCHEMICAL VISUALIZATION OF CATHEPSIN-D IN CULTURED-CELLS SUBJECTED TO OXIDATIVE STRESS

We describe a pre-embedding immunocytochemical method for visualizatio n of the lysosomal enzyme cathepsin D in cultured cells. The protein w as demonstrated at both light and electron microscopic levels by neutr al-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjuga ted to the antibodies. The best morphological preservation and the hig hest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium bor ohydride. Three cell types were used: human foreskin fibroblasts, hist ocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The r at heart myocytes were also exposed to the redox cycling substance nap hthazarin (5,8-dihydroxy-1,4-naphthoquinone) to induce oxidative stres s. This was done for such a short period of time that the cells initia lly did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was appa rent. This redistribution was followed by cell degeneration and, event ually, by cell death.