Hrp labeled goat anti rabbit secondary antibody

The proteins that isolated from MCF-7 cells by radioimmunoprecipitation (RIPA) Lysis Buffer (Beyotime, Shanghai, China) were then quantified using bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). After protein sample separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Danvers, MA, US). Samples were blocked using 5% skimmed milk for 1 h before overnight incubation with the primary antibodies against ABRACL (Sigma-Aldrich, cat. No. #HPA030217, 1:2500), MYBL2 (Abcam, ab12296, 1:2500), Ki67 (Abcam, ab92742, 1:5000), proliferating cell nuclear antigen (PCNA; Abcam, ab92552, 1:1000), matrix metallopeptidase 2 (MMP2; Abcam, ab92536, 1:1000), MMP9 (Abcam, ab76003, 1:1000), E-cadherin (Abcam, ab133597, 1:1000), Snail (Abcam, ab216347, 1:1000), Vimentin (Abcam, ab92547, 1:1000), N-cadherin (Abcam, ab76011, 1:5000) at 4°C. Prior to the rinse with phosphate buffer solution (PBS), HRP-labeled goat anti-rabbit secondary antibody (Beyotime, cat. No. A0208, 1:1000) was applied to foster the membranes at room temperature for 2 h. The protein blots were detected with the application of an enhanced chemiluminescent (ECL) Chemiluminescence Detection Kit (PromoCell, cat. No. PK-MB902-500-500) and subjected to analysis by Image Lab Software (Bio-Rad, Hercules, CA, US).

The paraffin-embedded femur tissues were sectioned at 3-μm thickness. After incubation with cleaved caspase-3 antibody (Cell Signalling Technology, Boston, MA, 1:100), LC3 antibody (Abcam, Cambridge, MA, 1: 200) and P62 antibody (Santa Cruz Biotechnology, CA, 1: 100) at 4 °C overnight, then incubated in HRP-labeled goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology, Inc., Jiangsu, China) for 30 min at 37 °C. After another 30-min incubation with Vectastain ABC-AP kit (Vector, CA), the specimens were rinsed and exposed to 3,3-diamino-benzidine (DAB) substrate for 3–10 min.

Resveratrol was purchased from Yixin Pharm, Inc. (Zhejiang, China); The triglyceride (TG) kit, total cholesterol (TC) kit, low density lipoprotein cholesterol (LDL-C) kit, high density lipoprotein cholesterol (HDL-C) kit, superoxide dismutase (SOD) kit, malonaldehyde (MDA) kit, free fatty acid (FFA) kit, reactive oxygen species (ROS) kit, glutathione (GSH) kit, glutathione peroxidase (GPx) kit, and the coomassie brilliant blue kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China); rabbit anti-mouse p47phox polyclonal antibody was purchased from LifeSpan Biosciences, Inc. (WA, USA); rabbit anti-mouse Sirt1 monoclonal antibody and the gp91phox polyclonal antibody were procured from Abcam, Inc. (Cambridge, UK); Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and p-HSL (Ser660) antibodies were purchased from Santa Cruz Biotech (Texas, USA); the primary antibodies including rabbit anti-mouse AMPK, p-AMPK (Thr172), FOXO1, and p-FOXO1 (Thr24) were purchased from Cell Signaling Technology (MA, USA); GAPDH antibody was obtained from Boster, Inc. (Wuhan, China); the BCA kit and HRP-labeled goat anti-rabbit secondary antibody were from Beyotime, Inc. (Jiangsu, China).

The prepared cochlear paraffin‐embedded sections were dewaxed, hydrated, and antigenically repaired, followed by blocking with 3% hydrogen peroxide and 3% bovine serum albumin (BSA) at room temperature for 30 min. The samples were then incubated with primary anti‐OPA1 (Abcam, ab157457), anti‐Ac‐SOD₂ (acetyl K68) (Abcam, ab137037), or acetylated‐lysine (CST, 9441S) at 4°C overnight, followed by incubation with an HRP‐labeled goat anti‐rabbit secondary antibody (Beyotime, A0208) at room temperature for 1 h. Staining with 3,3‐diaminobenzidine and counterstaining with hematoxylin were then conducted, followed by dehydration, mounting, and image collection. ImageJ was used to perform semi‐quantitative analyses of the staining results.