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Advisor(s)
Abstract(s)
Introduction: The implementation of methodologies capable of identifying apoptotic cells is a valuable tool in many biomedical studies. Much of the knowledge acquired about the mechanisms regulating apoptosis comes from observations in isolated cells; however, the properties shown by these assays do not always apply to the study of tissue samples. For detection of apoptosis in tissues, many investigators recommend the TUNEL method, nonetheless, other histochemical methodologies are available, and their choice must be carefully evaluated, taking into account the morphologic alterations which they intend to detect.
Objective: To share encountered difficulties in the implementation of three different methods for the detection of apoptotic cells in animal tissue.
Methods: Animal tissues, fixed in 10% neutral-buffered formalin and embedded in paraffin were submitted to immunohistochemical techniques using the TUNEL method (In situ Cell Death Kit, Roche) and polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology).
Results: After a long period of tests and costs, in which unspecific reactions, as well as several artifacts were obtained, the persistence to overcome these obstacles made all methods effective in the detection of apoptosis. Observed differences in immunolabeling were related to the type of tissues tested, which will be documented in the present study.
Conclusions: Immunohistochemical methods are an approach to consider in the detection of apoptosis in animal tissues. Sharing the challenging difficulties found in our work, might obviate many tests and costs in its implementation in other laboratory facilities.
Description
Keywords
apoptosis apoptotic events detection of apoptosis in tissues Immunohistochemical methods TUNEL method Bax Bcl-2
Citation
Garcia C, Cruz R, Mega A, Ortiz AL, Teixeira de Lemos E, Ferreira D, Reis F, Vala H (2013). Main difficulties encountered in implementation of techniques for apoptosis detection in animal tissues. XVIII Meeting of the Portuguese Society of Animal Pathology. International Meeting of Leporidae Pathology, University of Évora. Portugal, 9-10 Maio.