A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
1998 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 46, no 3, 411-418 p.Article in journal (Refereed) Published
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-naph-thoquinone) 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.
Place, publisher, year, edition, pages
1998. Vol. 46, no 3, 411-418 p.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-80993DOI: 10.1177/002215549804600316OAI: oai:DiVA.org:liu-80993DiVA: diva2:549669