Successful triple immunoenzymatic method employing primary antibodies from same species and same immunoglobulin subclass

Submitted: 16 March 2013
Accepted: 21 June 2013
Published: 16 September 2013
Abstract Views: 2537
PDF: 678
HTML: 2115
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Authors

Protocols for immunohistochemical (IHC) detection of multiple antigens in the same tissue sections have been developed using primary antibodies directly conjugated to different enzymes or fluorochromes, or ones that have been raised in different species, or from different immunoglobulin (Ig) classes or subclasses. For antibodies lacking such dissimilarities, very few proposals have been published with varying degrees of generalizability. In this report we present a successful triple IHC protocol engaging three unconjugated monoclonal primary antibodies raised in the same species and of the same Ig subclass. Compared to other methods, our results showed that denaturation of the preceding reaction complex by microwave heating, combined with additional suppression of enzyme activity, enabled the detection of all three reactions by using the same detection system, with no cross reaction observed. Moreover, expression patterns of each of the three antigens in the triple stained sections, was found to be similar to the pattern observed when single staining was performed. Unlike previous reports, no damage of targeted antigens or tissues did occur following this protocol. Furthermore, the contrast of the colors employed was investigated by computerized color deconvolution, and the three reactions products were successfully separated into three individual images that could be used for further objective quantification.

Dimensions

Altmetric

PlumX Metrics

Downloads

Download data is not yet available.

Citations

Supporting Agencies

Norwegian Dental Depots Fund, Gades legat, Norwegian Cancer Research Association and Bergen Medical Research Foundation.

How to Cite

Osman, T. A., Øijordsbakken, G., Costea, D. E., & Johannessen, A. C. (2013). Successful triple immunoenzymatic method employing primary antibodies from same species and same immunoglobulin subclass. European Journal of Histochemistry, 57(3), e22. https://doi.org/10.4081/ejh.2013.e22