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Journal of Histochemistry and Cytochemistry, Vol. 46, 411-418, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress

Karin Roberga and Karin Öllingera
a Division of Pathology II, Faculty of Health Sciences, Linköping University, Linköping, Sweden

Correspondence to: Karin Öllinger, Division of Pathology II, Faculty of Health Sciences, Linköping, Sweden.

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. (J Histochem Cytochem 46:411-418, 1998)

Key Words: immunocytochemistry, pre-embedding, cathepsins, ultrasmall gold, cultured cells, oxidative stress


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