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Journal of Histochemistry and Cytochemistry, Vol. 49, 355-368, March 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Optimization of Immunogold Labeling TEM: An ELISA-based Method for Rapid and Convenient Simulation of Processing Conditions for Quantitative Detection of Antigen

Ramandeepa, Kanak L. Dikshita, and Manoj Rajea
a Institute of Microbial Technology, Chandigarh, India

Correspondence to: Manoj Raje, Inst. of Microbial Technology, Sector 39A, Chandigarh 160114, India. E.mail: manoj@imtech.ernet.in

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, temperature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the results confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) of glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some distortion of cellular ultrastructure but better antibody binding activity compared to dehydration up to 100%. Postfixation or incorporation of tannic acid in the primary fixative caused almost total loss of activity, whereas antigen retrieval of osmium-postfixed material resulted in approximately 90–100% recovery. The sensitivity of detection of proteins by immunogold labeling electron microscopy depends on the retention of antibody binding activity during tissue processing steps, e.g., fixation and dehydration. Our study indicated that an ELISA-based screening method of various tissue processing procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM. (J Histochem Cytochem 49:355–367, 2001)

Key Words: antigen detection, quantitation, transmission electron microscopy, ELISA, tissue processing, antigen retrieval, artificial immunospecimen, immunogold labeling


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