Preembedding labeling with biotinylated antibodies and subsequent visualization of the biotin groups exposed on thin sections.
The feasibility of labeling cell membranes with biotinylated ligands and detecting the biotin groups on thin sections was investigated. Fixed retinal tissue was incubated with biotinyl- antiopsin . Half of the biotinyl-antibody labeled retinal tissue was incubated with avidin-ferritin (AvF) and embedded in Epon (preembedding reaction). The second half was embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Thin sections of this preparation were incubated with AvF to detect biotinyl-antibodies exposed by the sectioning (postembedding reaction). Biotin groups on the thin section surface could be readily visualized with AvF. Stereoscopic images demonstrated that the ferritin particles were localized only on the exposed surface of the thin section. The labeling was highly specific, with a very low background. Quantitative analysis was employed in order to determine the optimal reaction conditions for maximizing the labeling density with minimizing nonspecific binding. The possibility of using biotinylated molecules in the study of dynamic cellular events and for the subsequent intracellular localization of biotin on thin sections is suggested.
D000818:Animals; D000906:Antibodies; D001360:Avidin; D001710:Biotin / Q000032:analysis* / Q000276:immunology; D002462:Cell Membrane / Q000032:analysis; D005136:Eye Proteins / Q000032:analysis / Q000276:immunology; D005293:Ferritins; D006652:Histological Techniques; D008854:Microscopy, Electron; D010786:Photoreceptor Cells / Q000032:analysis; D011894:Rana pipiens; D012160:Retina / Q000032:analysis*; D012374:Rod Cell Outer Segment / Q000032:analysis; D017299:Rod Opsins
D000906:Antibodies; D005136:Eye Proteins; D017299:Rod Opsins; D001360:Avidin; D001710:Biotin; D005293:Ferritins