Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry.
We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.
D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D003102:Colloids; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005404:Fixatives; D006046:Gold; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D008862:Microsomes, Liver / Q000276:immunology* / Q000648:ultrastructure; D011506:Proteins / Q000032:analysis / Q000276:immunology*; D051381:Rats
D000941:Antigens; D003102:Colloids; D000939:Epitopes; D005404:Fixatives; D007106:Immune Sera; D011506:Proteins; D006046:Gold