K. Roberg et K. Ollinger, A PREEMBEDDING TECHNIQUE FOR IMMUNOCYTOCHEMICAL VISUALIZATION OF CATHEPSIN-D IN CULTURED-CELLS SUBJECTED TO OXIDATIVE STRESS, The Journal of histochemistry and cytochemistry, 46(3), 1998, pp. 411-418
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.