Rabbit anti p62 antibody

Osteoblasts were lysed in RIPA lysis buffer with protease inhibitors (Beyotime, Shanghai, China). Proteins were collected after centrifugation. The protein concentrations were determined by BCA Protein Assay Kit (Beyotime, China). The protein samples were subjected to SDS-PAGE (8–12%), and then transferred onto PVDF membranes (Millipore, Germany). After blocking with 5% skimmed milk (Bio-Rad, USA) for at least 1 h, the membranes were incubated with primary antibodies overnight at 4°C, including rabbit anti-LC3 antibody (1 : 1000, Novus), rabbit anti-Bnip3 antibody (1 : 2000, Abcam), rabbit anti-P62 antibody (1 : 1000, Abcam), rabbit anti-Beclin1antibody (1 : 1000, Abcam), mouse anti-caspase 3 antibody (1 : 500, Santa Cruz), rabbit anti-cleaved caspase 3 antibody (1 : 1000, CST), rabbit anti-Bcl-2 antibody (1 : 1000, CST), rabbit anti-Bax antibody (1 : 1000, CST) and mouse anti-β-actin antibody (1 : 8000, Tianjin Sungene Biotech). Depending on the origin of the primary antibodies, the secondary antibody (1 : 7000 – 1 : 10000) was added and incubated for 1 h at room temperature. The intensities of bands were quantified with Fusion software (VILBER LOURMAT, Germany). All the results were normalized to β-actin.

The fixed coronal sections (5-µm) of the hippocampus, aforementioned, were incubated with PBS containing 3% H₂O₂ at 37°C for 10 min to quench endogenous peroxidase activity, heated in antigen retrieval solution (EDTA, pH 8.0) at 90°C for 10 min, chilled in water and then immersed for 5 min in PBS at 37°C. Sections were then incubated with rabbit anti-Beclin-1 antibody (1:50; cat. no. ab62557; Abcam), rabbit anti-LC3A/B antibody (1:50; cat. no. 4108; Abcam) and rabbit anti-p62 antibody (1:50; cat. no. ab56416; Abcam) for 60 min at 37°C. Expression was then amplified with avidin biotin-peroxidase complex labelling and visualized using an IX71 inverted fluorescence microscope (magnification, ×400; Olympus Corporation). Data analysis was performed using ImageJ version 1.48 software (National Institutes of Health).