Tissue sections were prepared as described above. Following de-paraffinization, sections were washed for 10 min in distilled water. All subsequent procedures with tissue preparations were carried out at room temperature [21 (link),22 (link)]. Endogenous peroxidase activity was blocked with hydrogen peroxide (1:10 w/PBS, 10 min). Thereafter tissue sections were treated with proteinase K (catalog no. S3020; DAKO, Carpentaria, CA, USA) for 10 min. After two washes in PBS, tissue preparations were treated with TritonX for 15 minutes, rinsed in PBS and incubated with anti-Caspase 3 antibody (1:100 in PBS; cat no. ab17815 abcam). After two washes in PBS, preparations were treated with biotinylated goat anti-rabbit Ig (catalog no. HK336-9R, BioGenex). Following a 5-min wash in PBS, slides were incubated for 20 min in peroxidase-conjugated streptavidin (catalog no. HK330-9k, BioGenex). Caspase 3 cells were visualized using 3-amino-9-ethylcarbazole chromogen (catalog no. HK121-5K Liquid AEC, BioGenex) for 3–5 minutes for optimal staining. Preparations were counterstained with hematoxylin (catalog no. 7221; Richard-Allan Scientific, Kalamazoo, MI, USA) and mounted in Faramount (catalog no. S3025, DAKO).
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