Anti human bcl 2

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)]).

SDS-PAGE and Western blot analysis were performed according to the protocol described previously [16 (link)]. Briefly, cells were seeded in a 6-well plate, and after 24 h, treated with or without PEF-III (20 and 30 μg/mL) and incubated till 70–80% confluence. Lysates were prepared using lysis buffer (50 mM TrisCl, pH 7.8, 150 mM NaCl, 1% NP 40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride). The lysates were then resolved on SDS-PAGE and transferred into polyvinylidene difluoride (PVDF) membranes. Next, the membranes were blocked in skim milk (4%) and Tris-buffered solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h and incubated overnight at 4 °C with specific primary antibodies including anti-human Bcl-2 (Abcam, Tokyo, Japan), Bax (Cell Signaling Technology, Tokyo, Japan), p53 (Cell Signaling Technology), and β-actin (Cell Signaling Technology). After that, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 50 min and then washed with Milli-Q (3–4 times), after which bound antibodies were detected by chemiluminescence reaction using Immuno Star Zeta (Wako) and EZ capture MG (ATTO Corporation, Tokyo, Japan) according to the manufacturers’ protocols.

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)}.