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Journal of Histochemistry and Cytochemistry, Vol. 46, 1085-1090, September 1998, Copyright © 1998, The Histochemical Society, Inc.

TECHNICAL NOTE

Electron Microscopic Visualization of Receptor–mediated Endocytosis of DiI–labeled Lipoproteins by Diaminobenzidine Photoconversion

Nico P. Dantumaa, Marian A. P. Pijnenburga, Jacques H. B. Diederena, and Dick J. Van der Horsta
a Biochemical Physiology Research Group, Department of Experimental Zoology and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands

Correspondence to: Nico P. Dantuma, Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden..

We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells that had been incubated with DiI-labeled lipoproteins yielded a light- and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of DiI-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of DiI was not affected by aminotriazole. We conclude that specific staining of DiI-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here. (J Histochem Cytochem 46:1085–1089, 1998)

Key Words: photoconversion, diaminobenzidine, aminotriazole, DiI, electron microscopy, endocytosis, peroxisome, lipophorin, insect


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