Rabbit monoclonal anti active caspase 3

Digoxigenin-UTP-labeled riboprobes were synthesized according to the manufacturer’s instructions (Roche), and in situ hybridizations were performed as described previously [45 (link)]. The color reaction was conducted using the NBT/BCIP substrate (Roche). To minimize the variation between the control and experimental groups, we used embryos produced by a single pair of parents and always used the same number of embryos for the control and experimental groups. The groups were compared under precisely same experimental conditions at the same time, and color reactions were initiated and stopped at precisely the same time. For immunohistochemistry, zebrafish embryos were blocked in 5 % goat serum and incubated with a rabbit phospho-histone H3 antibody or rabbit monoclonal antiactive caspase-3 (1:200, Abcam). Goat antimouse IgG HRP or goat antirabbit IgG HRP (Roche) was used for detecting the primary antibodies, and DAB was used as a substrate for the secondary antibody-conjugated HRP (Amresco). The embryos were mounted in Vectashield mounting medium (Vector Laboratories, Inc.).

Caco-2 cells were harvested, washed with PBS, fixed in 4% formaldehyde in PBS for 15 min and permeabilized with 0.3% Triton-X100 in PBS for 1 h. Two percent bovine serum albumin (BSA) was used to block the non-specific binding sites. The cells were then incubated overnight with mouse anti-ZO-1 (1:100) and rabbit anti-occludin antibody (1:100), or rabbit monoclonal anti-active caspase-3 (1:100; Abcam, Cambridge, UK) and further incubated in the dark with the appropriate secondary antibody (fluorescein isothiocyanate conjugated anti-rabbit or Texas red conjugated anti-mouse). The cells were analyzed using a microscope (Nikon Eclipse 80i), and images were captured by a high-resolution digital camera (Nikon Digital Sight DS-U1). Appropriate negative controls were done by omitting primary or secondary antibodies.

Apoptosis proteins, caspase-3, and PARP were assessed within cell lysate after treatment with TQ, PTX, and their combination to confirm cell death propagation via apoptosis. Briefly, cells were treated with the pre-determined IC₅₀ values of PTX and TQ for 24 h and 48 h. Cell lysates were extracted using an RIPA-buffer and electrophoresed using SDS-PAGE (10%) and then transferred to PVDF-membrane. Caspase-3 and cleaved PARP-1 proteins were detected using rabbit monoclonal anti-active caspase-3 and rabbit monoclonal anti-PARP (Abcam Inc., Cambridge Science Park, Cambridge, UK). Bands were visualized using HRP-conjugated anti-rabbit secondary antibodies (Abcam Inc., Cambridge Science Park, Cambridge, UK).