A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques.
The purpose of this study was to compare the level of immunogold labelling of both osmicated and non-osmicated epoxy sections when subjected to different antigen retrieval, etching and incubation temperature for the antibodies. Pure IgG protein gels were produced by glutaraldehyde fixation, eventually postfixed with 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were antigen retrieved in citrate solution at 95 or 144 degrees C and eventually etched with NaIO4. Immunogold labelling with anti-IgG was performed at 4 degrees C overnight or at 60 degrees C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 degrees C and incubated with primary antibodies at 60 degrees C than when heated at 95 degrees C, etched with NaIO4 and incubated with primary antibodies at 4 degrees C. Osmium-fixed IgG-gels antigen retrieved at 144 degrees C and incubated with anti-IgG at 60 degrees C showed more labelling than sections of non-osmicated gels heated at 95 degrees C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 degrees C for 1 h than at 4 degrees C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.
Brorson
Sverre-Henning
SH
Department of Pathology, Ullevål University Hospital, Kirkeveien 166, 0407 Oslo, Norway. s.h.brorson@labmed.uio.no
D000818:Animals; D000936:Antigen-Antibody Complex; D000941:Antigens / Q000032:analysis; D004853:Epoxy Resins; D006358:Hot Temperature; D006801:Humans; D007074:Immunoglobulin G; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron; D016613:Plastic Embedding / Q000379:methods*; D011817:Rabbits
D000936:Antigen-Antibody Complex; D000941:Antigens; D004853:Epoxy Resins; D007074:Immunoglobulin G