An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.
We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.
Department of Biology, University of Virginia, Charlottesville 22901.
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Berryman
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Rodewald
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D005404:Fixatives; D006056:Golgi Apparatus / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods*; D007583:Jejunum / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D010179:Pancreas / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D011961:Receptors, Fc / Q000032:analysis; D013194:Staining and Labeling; D014021:Tissue Preservation / Q000379:methods
D005404:Fixatives; D011961:Receptors, Fc