On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue.
On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.
Johnson
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Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461.
Bettica
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D003599:Cytoskeleton / Q000648:ultrastructure*; D004853:Epoxy Resins; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006652:Histological Techniques; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009457:Neuroglia / Q000378:metabolism / Q000648:ultrastructure*
D004853:Epoxy Resins; D005904:Glial Fibrillary Acidic Protein