A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques

Abstract

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 °C and eventually etched with NaIO 4. Immunogold labelling with anti-IgG was performed at 4 °C overnight or at 60 °C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 °C and incubated with primary antibodies at 60 °C than when heated at 95 °C, etched with NaIO 4 and incubated with primary antibodies at 4 °C. Osmium-fixed IgG-gels antigen retrieved at 144 °C and incubated with anti-IgG at 60 °C showed more labelling than sections of non-osmicated gels heated at 95 °C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 °C for 1 h than at 4 °C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.