Anti human bax

After fixation of the islets in 10 % buffered formalin, they underwent embedding in agar, and the second embedding in paraffin was performed. The blocks were sectioned at 5 µm. Next, de-paraffinization was done for immunocytochemistry analysis. The slides were incubated with primary antibodies including anti-human insulin, anti-human BAX, anti-human BCL2, and anti-human caspase-3 (All from Abcam, USA), at 4 °C overnight. After washing, the secondary antibody (Abcam, USA) was used for the detection. The nuclei were stained by hematoxylin. The H-score was applied to estimate the positive level of the islets, as the following formula: H score = 1 × (% light staining) + 2 × (% moderate staining) + 3 × (% strong staining) (Detre et al., 1995[2 (link)]).

The islet groups were fixed in 4% paraformaldehyde and embedded in low melting agarose (Sigma, Germany). After dehydration process, the second embedding was performed in paraffin, cut in 5 μm sections, deparaffinised, and surveyed by immunocytochemistry. Immuno-labelling was done with primary antibodies specific to anti-human Bax (dilution 1:50; Abcam, France), anti-human Bcl-2 (dilution 1:250; Abcam, France), anti-human active Caspase-3 (dilution 1:50; Abcam, France), anti-human HIF-1α (dilution 1:50; Medaysis, USA), and anti-human p53 (dilution 1:50; Dako, Canada). All of these primary antibodies were detected by HRP- secondary antibody (dilution 1:200; Abcam, France). Finally, the samples were colored with a chromogen 3,3′- diaminobenzidine (DAB) (Dako, Canada) and counterstained with haematoxylin. The brown stained areas were scored based on the H-score method with following formula: H score = 1 × (% mild staining) + 2 × (% moderate staining) + 3 × (% strong staining)^{48 (link)}.