Post-embedding immunolabelling. Some effects of tissue preparation on the antigenicity of plant proteins

Abstract

The sensitivity of detection of proteins by immunolabelling embedded and sectioned material depends upon retention of antigenicity during tissue processing. Losses in pea seed storage protein antigenicity have been assessed using a solid phase radioimmunoassay. Storage proteins adsorbed to wells of microtiter plates were treated with the various "tissue preparation" steps and reacted with 125I-antibodies to the storage proteins. The glutaraldehyde or formaldehyde fixation steps caused approximately 60% and 30% loss in antigenicity, respectively. With each subsequent preparative step, losses accumulate and following treatment with Spurr's epoxy resin reached approximately 85% and 90%, respectively. However, if Lowicryl K4M, a methacrylate-acrylate embedding medium was used, losses were retained at approximately 70% and 60%, respectively. These observations have been verified on sections of embedded material using two labelling procedures, with protein A-gold and ferritin as the markers.