Tissue fixation effects on immunohistochemical staining of caspase-3 in brain tissue.
Fixation methods for tissue often vary amongst clinical and research laboratories. To evaluate the effects of fixation method on studies of brain tissue, we examined immunohistochemical outcomes amongst 2 fixatives, 4 caspase-3 antibodies, and 2 species (human infants and piglets). Fixatives were 10% neutral buffered formalin (NBF) or 10% NBF and glacial acetic acid (FAA). Antibodies for caspase-3 were commercially obtained and included 2 for active caspase-3, and 2 for procaspase-3 (CASP3 and CPP32). Immunohistochemical staining of caspase-3 varied with fixation method, with the greatest effect of fixation method observed for the active caspase-3 antibodies and this effect was present in both species. In NBF-fixed tissue, active caspase-3 immunoreactivity was only visible microscopically, and was specific to neuronal cell bodies. In FAA-fixed tissue, active caspase-3 immunoreactivity was visible macroscopically, and predominantly present in fiber tracts and fasciculi compared with neuronal bodies. Fixation and species differences were also identified for the procaspase-3 antibodies, CASP3 and CPP32, where FAA-fixed pig tissue showed abundant staining of blood vessels that were not observed in the NBF-fixed pig tissue or in the human tissue. This study characterizes differences in immunohistochemical outcomes using commercially available antibodies for caspase-3, according to tissue fixation method and species.
Machaalani
Rita
R
Department of Medicine, , The University of Sydney, NSW 2006, Australia. ritam@med.usyd.edu.au
Radford
Jane L
JL
Waters
Karen A
KA
D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000201:enzymology*; D053148:Caspase 3 / Q000032:analysis*; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008297:Male; D013194:Staining and Labeling; D013552:Swine; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D005557:Formaldehyde; D053148:Caspase 3; D019342:Acetic Acid