Anti rabbit peroxidase conjugated secondary antibodies

Supernatant treated HT-22 cells were harvested for obtaining total cellular extracts, and the protein isolation procedure and immunoblotting steps were performed according to standard procedure. After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies against cleaved caspase-3 (Abcam, USA), at 1:1,000 dilutions. After extensive washes with 0.1% PBS-Tween, blots were incubated with the Anti-Rabbit peroxidase-conjugated secondary antibodies (Vector Laboratories, USA). The blots were processed for development using chemiluminescence reagent (Millipore, USA). The images were captured and analyzed using the Chemigenius bioimaging system (Syngene, United Kingdom). β-actin antibody (Sigma, USA) at 1:10,000 dilution was used as loading control.

HT22 cells under different treatment conditions were harvested for obtaining total cellular extracts, and the protein isolation procedure and immunoblotting steps were performed according to standard procedure. After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies against cleaved caspase 3 (Abcam, United States), Cleaved Caspase 8 (Cell signaling, United States), FADD (Sigma, United States), p-PLC-γ (Cell signaling, United States), p-p38(Cell signaling, United States), CHPV (a kind gift by Bharat Biotech International Limited, Hyderabad, India) at 1:1,000 dilutions. After extensive washes with 0.1% PBS-Tween, blots were incubated with the Anti-Rabbit peroxidase-conjugated secondary antibodies (Vector Laboratories, United States). The blots were processed for development using chemiluminescence reagent (Millipore, United States). The images were captured and analyzed using the Uvitec Cambridge using NineAlliance software (Uvitec, United Kingdom). β-actin antibody (Sigma, United States) at 1:10,000 dilution was used as loading control.