Anti cyt c

The chemicals utilized during study: ammonium sulphate, 2,4-dinitro-phenyl-hydrazine, 1-chloro-2,4-dinitrobenzene, ethylenediaminetetraacetic acid, 5,5-di-thio-bi(2-nitrobenzoic acid), N-ethylmaleimide, nitro blue tetrazolium, reduced nicotinamide adenine dinucleotide, potassium dihydrogen phosphate, phenazinemethosulphate, sodium pyrophosphate, reduced glutathione, sodium azide, thiobarbituric acid, 5-thio-2-nitrobenzoic acid and trichloro acetic acid were procured from Sisco Research Laboratory, India. Bovine serum albumin, Bradford reagent and Pb-acetate were purchased from Sigma-Aldrich, St. Louis, USA. All primary antibodies (produced in rabbit) viz. anti-Bcl-2 (dilution 1:1000), anti-Bad (dilution 1:3000), anti-Cyt C (dilution 1:1000), anti-Apaf-1 (dilution 1:1000), anti-caspase 3 (dilution 1:1000), anti-caspase 8 (dilution 1:1000), anti-caspase 9 (dilution 1:1000), anti-Fas (dilution 1:2000), anti-Bid (dilution 1:1000) and anti-actin (dilution 1:3000) for immunoblotting were purchased from Sigma-Aldrich Chemical Company St. Louis, USA. Appropriate HRP conjugated secondary antibody (dilution 1:3000) produced in goat was also purchased from Sigma-Aldrich Chemical Company St. Louis, USA. Methanol, formic acid, acetic acid and acetonitrile were obtained from Merck, India.

Immunocytochemistry was performed and modified according to Iida’s study. Briefly, SH-SY5Y cells were washed with PBS three times, fixed with PBS containing 4% (wt/vol) paraformaldehyde for 15 min, and then permeabilized with 0.5% (wt/vol) Triton X-100 in PBS for 20 min. Immunocytostaining was performed with anti-Akt (1:200), anti-P-Akt (1:200), anti-PI3K (1:200), anti-P-PI3K (1:200), anti-BAD (1:200), anti-Bax (1:100), anti-Bcl-2 (1:100), anti-Cytc (1:200), anti-GSK3β (1:200), anti-p53 (1:100), anti-NGF (1:200), and anti-TrkA (1:200) antibodies (Sigma). After the nonspecific reaction was blocked with PBS containing 10% (wt/vol) bovine serum albumin (BSA), the cells were incubated with the primary antibody in PBS overnight, washed with PBST, and incubated with the second antibody (1:200) in PBST for 1 h. After the samples were washed with PBS three times, they were embedded in DAPI for 5 min and then washed with PBST four times. The images were obtained using an Olympus microscope (Shanghai, China). The mean fluorescence intensity was calculated by Image-Pro software (Meyer, TX, USA).