Use of uranyl acetate en bloc to improve tissue preservation and labeling for post‐embedding immunoelectron microscopy

Abstract

AbstractWe have developed a protocol for post‐embedding immunoelectron microscopy that utilizes uranyl acetate, en bloc, as a secondary tissue fixative. Squirrel and cat retinas were fixed in 1% paraformaldehyde‐1% glutaraldehyde for one hour. Secondary fixation was by 2% uranyl acetate, en bloc, (1 hour) during tissue dehydration. The tissue was embedded in LR White or Lowicryl K4M resin. Post‐embedding immunoelectron microscopy (indirect immunogold) was performed on thin sections with antibodies to four different classes of proteins (filamentous, cytoplasmic, membrane, and extracellular matrix). The sections were then stained sequentially on drops of uranyl acetate and lead citrate, and by vapors of osmium tetroxide. Uranyl acetate fixation and/or staining of the sections by osmium tetroxide was omitted from the control experiments. Differences after secondary fixation with uranyl acetate and staining of the thin sections with osmium tetroxide were better overall preservation and enhanced contrast of the extracellular matrix, membranes, cytoplasm, and DNA. Antigenicity, as evidenced by the immunolabeling of the four proteins, was retained. Quantitation of the immunolabeling for the cytoplasmic and membrane proteins revealed significantly increased labeling densities in tissue postfixed with uranyl acetate. The improved tissue preservation and immunolabeling of proteins indicate that secondary fixation with uranyl acetate can be a valuable addition to post‐embedding immunocytochemistry.