A pre-embedding technique for immunocytochemical visualization of cathepsin D in cultured cells subjected to oxidative stress.
We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest 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 borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5, 8-dihydroxy-1,4-naphthoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially 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 apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.
Roberg
K
K
Division of Pathology II, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
Ollinger
K
K
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D016403:Lymphoma, Large B-Cell, Diffuse / Q000201:enzymology; D008297:Male; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016610:Tissue Embedding / Q000379:methods*; D014407:Tumor Cells, Cultured
D002402:Cathepsin D