Anti p47 phox sc 17844

After treatments, the cells were suspended in 2 ml of saline A and incubated for 30 min in 35-mm tissue culture dishes containing an uncoated coverslip. Under these conditions, cells rapidly attach to the coverslip. In some experiments the cells were incubated for 5 min with the cell-permeable DNA dye (Hoechst 33342, 10 μM) prior to the end of incubation to stain the nucleus. The cells were then fixed for 1 min with 95% ethanol/5% acetic acid, washed with PBS and blocked in PBS containing bovine serum albumin (2% w/v) (30 min at room temperature).
The cells were subsequently incubated with rabbit polyclonal anti-P47 phox (sc-17844) (1:100 in PBS supplemented with 2% bovine serum albumin; Santa Cruz Biotechnology Inc. USA), rabbit polyclonal anti-Nrf2 (1:50 in PBS containing 2% bovine serum albumin; Santa Cruz Biotechnology) or monoclonal anti-cytochrome c antibody (1:100 in PBS containing 2% bovine serum albumin; Santa Cruz Biotechnology) stored for 18 h at 4° C, washed and then incubated for 3 h in the dark with fluorescein isothiocyanate (Santa Cruz Biotechnology)-conjugated secondary antibody diluted 1:100 in PBS. Stained cells were captured with a fluorescence microscope and the resulting images were processed for fluorescence determination as described above.