Caspase 8 polyclonal antibody

Colon tissues were fixed and embedded with formalin, and 4 μm thick sections were cut. After deparaffinizing, rehydrating, 0.1 M citrate buffer was used for antigen recovery. These slices were then blocked the non-specific binding following soaking in goat serum for 30 min. They were then incubated with primary Caspase-3 antibody (1:1,000, “9,662”; Cell Signaling Technology, Danvers, United States), Caspase-8 Polyclonal antibody (1:100, 13423-1-AP; Proteintech, Wuhan, China). After three 5-min washes with PBS, the second antibody was put on slides and incubated for 30 min. Then DAB and hematoxylin were used to stain, respectively. Mean of IOD was evaluated using the Image-Pro (^®) Plus software.

Protein extraction and concentration determination were performed as above. SDS-PAGE was used to separate the proteins in the sample by molecular weight in the gel, and then the proteins were transferred to PVDF membranes, which were blocked with 2.5% skim milk for 1 h at room temperature, and then the PVDF membranes were incubated with primary antibodies overnight. The primary antibodies used in this experiment: ZO-1 Polyclonal Antibody (1:1,000, “abs131,224”; Absin, Shanghai, China), Occludin Polyclonal Antibody (1:1,000, “abs136,990”; Absin, Shanghai, China), Caspase-3 antibody (1:1,000, “9,662”; Cell Signaling Technology, Danvers, United States), Caspase-8 Polyclonal antibody (1:500, 13423-1-AP; Proteintech, Wuhan, China). After the primary antibody incubation, membranes were washed three times with TBST for 10 min each. Then they were incubated with HRP-conjugated secondary antibody diluted in 2.5% skim milk for 1 h at room temperature. Signals were visualized through the electrochemiluminescence (ECL) detection system. Protein fluorescence density and relative expression were evaluated by Image J software (NIH, Bethesda, United States).