A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer |
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Authors: | Joshua R Edwards Evangelos A Diamantakos Jacob D Peuler Peter C Lamar and Walter C Prozialeck |
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Institution: | (1) Department of Pharmacology, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA |
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Abstract: | Background Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells
in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves
perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated
with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg2+, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule.
Twenty-four h after the administration of Hg2+, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was
then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with
-20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined
from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium
homodimer labeling. |
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