Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bc1-2 protein in formaldehyde-fixed, paraffin-embedded sections.
Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up t o 100 degrees C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130 degrees C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.
Hoetelmans
R W M
RW
Department of Medical Information, GSK, Zeist, The Netherlands. Rob.W.Hoetelmans@GSK.com
van Slooten
H J
HJ
Keijzer
R
R
van de Velde
C J H
CJ
van Dierendonck
J H
JH
D000818:Animals; D005557:Formaldehyde; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry; D008872:Microwaves; D016612:Paraffin Embedding; D011312:Pressure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D051381:Rats; D016707:Tissue Fixation / Q000379:methods*
D019253:Proto-Oncogene Proteins c-bcl-2; D005557:Formaldehyde