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

Micron : the International Research and Review Journal for Microscopy
Sverre-Henning Brorson

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 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...Continue Reading

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Citations

Aug 18, 2012·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Murray C KillingsworthC Soon Lee
Dec 10, 2008·Journal of Immunological Methods·Fabio D'AmicoFranca Stivala
Jan 2, 2007·Progress in Histochemistry and Cytochemistry·Shuji Yamashita
Oct 12, 2012·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Qianyue JinGaiping Zhang
May 23, 2014·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Shuji Yamashita, Yasunori Okada

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