Optimization of immunogold labeling TEM: An ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen

We developed an ELISA-based method for rapid optimization of various tissue processing parameters in immunogold labeling for electron microscopy. The effects of aldehyde fixation, tannic acid, postfixation, dehydration, tempe rature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the r esults confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) o f glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some di stortion of cellular ultrastructure but better antibody binding activity co mpared to dehydration up to 100%. Postfixation or incorporation of tannic a cid in the primary fixative caused almost total loss of activity, whereas a ntigen retrieval of osmium-postfixed material resulted in approximately 90- 100% recovery. The sensitivity of detection of proteins by immunogold label ing electron microscopy depends on the retention of antibody binding activi ty during tissue processing steps, e.g., fixation and dehydration. Our stud y indicated that an ELISA-based screening method of Various tissue processi ng procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM.