Anti cleaved caspase 3

IHC was performed on 5-μm-thick formalin-fixed paraffin-embedded slices. After deparaffinization, rehydration, antigen retrieval and endogenous peroxidase inhibition, the samples were incubated with anti-NSD2 (Abcam), anti-TUNEL (Roche) and anti-cleaved caspase 3 (Servicebio) antibodies at 4 °C for 8–10 h. After the secondary antibody was applied, diaminobenzidine (DAKO) solution was used as a chromogen. Furthermore, haematoxylin (Sigma) staining was performed to identify nuclei. Images were acquired using a microscope (Leica DM 4000B).

Western blot analysis was carried out to detect the protein expressions of apoptosis-related indicators, including Bax, Bcl-2, Caspase-3, cleaved-Caspase-3, and members of the AMPK/SIRT1/PGC-1α pathway, as described previously (Han et al., 2020 (link)). Briefly, total protein was extracted, separated on SDS-PAGE gels (10%, Servicebio, China), transferred onto a PVDF membrane (Servicebio, China). The membranes were blocked with 5% (W/V) skimmed milk TBST (pH 7.3) for 1 h at 37°C and incubated overnight in 4°C with the major antibody of anti-Bcl-2 (dilution: 1:100,000) (Servicebio, China), anti-Bax (dilution: 1:1,000) (Affinity, United States), anti-Caspase-3 (dilution: 1:1,000) (Servicebio, China), anti-cleaved-Caspase-3 (dilution: 1:1,000) (Servicebio, China), anti-AMPK (dilution: 1:1,000) (BIOSS, United States), anti-Sirt1 (dilution: 1:1,000) (San Ying, China), and anti- PGC-1α (dilution: 1:1,000) (Servicebio, China). Then the immobilized primary antibody conjugated with the secondary antibody (dilution: 1: 5,000) (Servicebio, China) in TBST for 30 min at room temperature. After a thorough washing with TBST, proteins were visualized with ECL (Servicebio, China). Quantification of protein expression was performed using the analysis software of Alpha.