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

Ramandeep, K and Dikshit, Kanak L and Raje, Manoj (2001) Optimization of immunogold labeling TEM. An ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 49 (3). pp. 355-68. ISSN 0022-1554

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Abstract

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.

Item Type: Article
Additional Information: Copyright of this article belongs to Histochemical Society
Uncontrolled Keywords: antigen detection; antigen quantitation; transmission electron microscopy; nonspecific binding artificial immunospecimen ELISA blocking signal-to-noise ratio immunogold labeling
Subjects: Q Science > QD Chemistry
Depositing User: Dr. K.P.S.Sengar
Date Deposited: 04 Jan 2012 14:57
Last Modified: 29 Mar 2012 09:50
URI: http://crdd.osdd.net/open/id/eprint/302

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