Background. Immunohistochemistry (IHC) is the localization of antigens in different tissues using specific primary and secondary antibodies. IHC is widely used in basic research and surgical pathology, both in human and animal models. Frozen and fixed tissues can be used but fixation and paraffin embedding offer the best option for preserving the specimen morphology. Unfortunately, the most common fixative (10% buffered formalin) may alter the biochemistry of the proteins and mask antigens. For this reason, antigen retrieval is required to allow antigen-antibody binding and different types of digestive enzymes or heat-induced methods can be used. Murine models have always been a challenge for IHC due to low sensitivity of mouse antibodies binding mouse tissues and limited availability of immunohistochemical reagents for formalin fixed tissues. In fact, most of the mouse-specific antibodies are only functional on frozen tissues but the quality of frozen sections is not good enough for morphological evaluation. Aim. To propose a new and sensitive approach, based on heat-antigen retrieval, for IHC on formalin fixed-paraffin embedded murine tissues in order to provide a useful panel of antibodies for immunology research. Methods. Tissues: murine colon, small bowel, lung, spleen. Fixative: 10% buffered formalin (24 hour at room temperature). We used a pressure cooker specifically designed for antigen retrieval (temperature of 125 C degrees and 20 psi, unmasking buffer at pH 6.00 which turns during boiling to 7.00) and tested a panel of 31 antibodies to identify leukocytes, endothelial and epithelial cells, cytoskeleton molecules, proliferation markers, and cytokines. These antibodies either cross-reacted with murine antigens or were mouse-specific. Immunohistochemical staining was carried out on an automated immunostainer. We compared several dilutions of the antibodies, and used various detection systems. Results. See table 1. Overall, 21 of the tested antibodies showed specific positivity (highlighted in white), 4 antibodies failed to work (highlighted in red) and 6 produced a strong background which made it difficult to analyze the results (highlighted in yellow). The morphology of the cells and tissues was entirely preserved in all of the samples. In table 1 we reported the technical characteristics of the antibodies and our results in terms of: recommended concentration, recommended detection system for each antibody. Conclusions. We demonstrated the useful application of an innovative method for immunohistochemical analysis on formalin fixed murine tissues: the decloaking chamber. This method will guarantee to deliver a clear and specific staining compared to other well recognized techniques
A new and sensitive approach for immunohistochemical analysis on formalin fixed murine tissues / D. Russo, M. Nebuloni, F. Pasqualini, G. Tasso. ((Intervento presentato al convegno nazionale SIAPEC-IAP tenutosi a Palermo nel 2011.
|Titolo:||A new and sensitive approach for immunohistochemical analysis on formalin fixed murine tissues|
NEBULONI, MANUELA (Secondo)
|Data di pubblicazione:||2011|
|Settore Scientifico Disciplinare:||Settore MED/08 - Anatomia Patologica|
|Citazione:||A new and sensitive approach for immunohistochemical analysis on formalin fixed murine tissues / D. Russo, M. Nebuloni, F. Pasqualini, G. Tasso. ((Intervento presentato al convegno nazionale SIAPEC-IAP tenutosi a Palermo nel 2011.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|