Immunofluorescence for Bax, Bcl-2, active Caspase-3, & cleaved PARP-1 in the kidney sections using confocal microscopy

The kidney tissue was fixed in 10% formalin, embedded in paraffin wax and 5 μm thick sections were cut by using the rotatory microtome, Lieca RM2125 RT (Leica Biosystems Nussloch GmbH, Nussloch, Germany) and the Immunofluorescence of Bax, Bcl-2, active Caspase-3 and cleaved PARP-1 was performed in a two-step procedure as mentioned elsewhere [35]. Briefly, kidney sections were deparaffinized in xylene and rehydrated in graded series of alcohol. Heat induced antigen retrieval was performed using 1 M citrate buffer (pH 6) in a microwave oven at 1000 W (1–2 min). After treating with blocking solution for 2 h at room temperature (5% HIGS- Heat Inactivated Goat serum), different sections were incubated with different antibodies Bax(dilution1:25), Bcl-2(dilution 1:50), active Caspase-3 (dilution 1:50) and cleaved PARP-1 (dilution 1:20) for 24 h in humid chamber. Sections were then washed with TBS (3 X 5 min.) and incubated with rabbit anti-mouse IgG H&L (conjugated with FITC) and goat anti-rabbit IgG H&L (conjugated with TRITC), (1:200) for 3 h. at room temperature in dark. After incubation, sections were again washed with TBST(tris bufferes saline with tween-20) (3 X 5 min.) and two drops of the fluorescent media (0.5% N-propyl gallate + 20 mM Tris in 90% glycerol + DAPI) were applied on the sections. Coverslips were applied and then sealed with nail polish after ensuring the spread of the mounting media over all the sections without any bubble formation). Nuclei were counterstained with DAPI (1 μg/10 ml PBS).