Mouse anti caspase 3

The liver tissue slices were incubated in proteinase K (20 μg/ml) for 5 min for proteolysis and were blocked with 10% normal goat serum for 4 h at room temperature. Then, the samples were incubated for 72 h in a 4℃ humidified chamber with the following primary antibodies: mouse anti-PPARγ (1:50, Santa Cruz, CA), mouse anti-p-mTOR (1:50, Santa Cruz, CA), mouse anti-glutathione peroxidase 4 (GPx4) (1:100, Santa Cruz, CA), mouse anti-8-OHdG (1:100, Santa Cruz, CA), mouse anti-caspase 3 (1:100, Santa Cruz, CA), mouse anti-p-IkB (1:250, Santa Cruz, CA), mouse anti-ERK (1:100, Santa Cruz, CA), and mouse anti-p-JNK0 (1:100, Santa Cruz, CA). Biotinylated goat anti-mouse IgG (1:100, Abcam, Cambridge, UK) secondary antibodies were incubated for 48 h in a 4℃ humidified chamber. Finally, the samples were incubated with the avidin biotin complex solution (ABC kit, Vector Laboratories Inc., Burlingame, CA) for 1 h at room temperature. The immune-reactivity was visualized in a 50-mM Tris−HCl solution (pH 7.4) containing 0.05% 3,3´-diaminobenzidine and 0.01% H₂O₂, followed by counterstaining with hematoxylin.

Immunohistochemical staining was performed as described previously, with few modifications.25 (link) After deparaffinization, slides were processed for the antigen-retrieval step (enzymatic method), then washed with PBS. The endogenous peroxidase was quenched by applying 3% H₂O₂ in methanol for 10 minutes. Slides were incubated with 5% normal goat serum containing 0.1% Triton X-100. After blocking, slides were incubated overnight with mouse anti-JNK (pJNK), mouse anti–caspase 3, mouse anti-TNFα, and mouse anti-COX2 antibodies (dilution 1:100, Santa Cruz Biotechnology). The following morning, after being washed with 0.1 M PBS, slides were incubated in biotinylated secondary antibody (dilution 1:50) according to the origin of the primary antibody and serum used. Following secondary antibody treatment, slides were incubated with the ABC Elite kit for 1 hour in a humidified chamber, then washed with 0.1 M PBS, stained in 3,3′-diaminobenzidine peroxidase solution, washed with distilled water, dehydrated in a graded ethanol series, fixed in xylene, and coverslipped in mounting medium. Immunohistochemical TIF images were captured with light microscopy.

Following schisandrin B treatment for 5 days, rats (n=6) were sacrificed and spinal cord samples were collected. The spinal cord was homogenized with liquid nitrogen and radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The homogenate was centrifuged at 12,000 × g for 10 min at 4°C and analyzed using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). An equal quantity (50 µg) of protein was separated by 8–10% SDS-PAGE, then transferred onto nitrocellulose filter membranes. Membranes were blocked with 5% skim milk powder in TBST for 1 h at 37°C and detected using the following primary antibodies: Mouse anti-caspase-3 (catalog no. sc-22139; dilution, 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); p-p53 expression (catalog no. sc-12904-R; dilution, 1:1,000; Santa Cruz Biotechnology, Inc.); or mouse anti-β-actin (catalog no. BB-2116-1;dilution, 1:5,000; BestBio Inc., Shanghai, China) at 4°C overnight. Proteins of interest were detected with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (catalog no. sc-2005; dilution, 1:5,000; Santa Cruz Biotechnology, Inc.) Proteins were observed using an enhanced chemiluminescence detection ECL kit (BestBio Inc.) and quantified using imaging software (BioSens Digital Imaging 5; Shanghai Bio-Tech Inc., Shanghai, China).