Localization of α(2u)-globulin within protein droplets of male rat kidney: Immunohistochemistry using perfusion-fixed, GMA-embedded tissue sections

Abstract

We investigated the light microscopic subcellular localization and quantitation of α(2u)-globulin (α(2u)G) in male rat kidney after 2,2,4-trimethylpentane exposure using a monoclonal antibody to α(2u)G. Slices of perfusion-fixed kidney were cold-processed in glycolmethylacrylate and the antigen localized after an avidin-biotin-horseradish peroxidase procedure with Hanker-Yates reagent as the chromagen. Light microscopic examination revealed resolution comparable to low-magnification electron microscopy, with excellent morphological detail of tissue architecture, including subcellular localization of α(2u)G within lysosomes of P2 segment cells of the proximal tubule epithelium. The anatomical relationship between α(2u)G and TMP-induced protein droplets observed after staining with Lee's methylene blue-basic fuchsin was studied using serial sections. Image analysis of selected P2 segments in treated and control rats revealed a high correlation between subcellular localization of α(2u)G and protein droplet deposition in the cytoplasm of P2 segment cells of the proximal tubule epithelium. Quantitative morphometry of α(2u)G-stained proximal tubule epithelium 72 hr after treatment with 50 mg/kg 2,2,4-trimethylpentane p.o. demonstrated a 1.5- to 2-fold increase in staining area of tubules from treated rats compared with controls. Similar increases of Lee's methylene blue-basic fuchsin-stained protein dropletes were also observed, but quantitative morphometry of the protein droplets was technically more difficult owing to a lower staining contrast between droplets and the surrounding cytoplasm. This immunohistochemical procedure provides a valuable technique for further studies on the pathological role of α(2u)G in protein droplet nephropathy of male rats induced by many environmental chemicals, and demonstrates the value of cold glycolmethacrylate processing to improve morphological detail.