Pre-embedding immunogold localization of antigens in mammalian brain slices.
The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have been developed since the introduction of colloidal gold to immunocytochemistry. The two most widely used techniques, however, are based on transmission electron microscopy and consist of either immunolabeling before the specimens are embedded in resin (pre-embedding immunogold labeling) or immunolabeling after embedding in resin (post-embedding immunogold labeling). The following protocol describes a pre-embedding procedure that gives reliable results with all antibodies that produce adequate staining as observed with a light microscope. This procedure results in almost perfect preservation of the ultrastructure. The procedure employs thick sectioning using a vibratome, permeabilization of membranes with Triton X-100, and immunolabeling with fluorescently conjugated Nanogold antibodies, followed by gold enhancement and embedding for electron microscopy. We also discuss some limitations inherent to pre-embedding immunogold labeling.
Schikorski
Thomas
T
Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico, USA.
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000941:Antigens / Q000032:analysis* / Q000276:immunology / Q000648:ultrastructure; D001921:Brain / Q000276:immunology* / Q000648:ultrastructure*; D001923:Brain Chemistry; D017979:Gold Colloid / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry; D051379:Mice; D016610:Tissue Embedding / Q000379:methods*
D000906:Antibodies; D000941:Antigens; D017979:Gold Colloid