Ab32047

Western blotting was performed as described previously [20 (link)]. Briefly, the brain protein samples were prepared using Ripa Lysis buffer (Bio-Rad, CA), and equal amounts of protein were run on an SDS-PAGE gel. After being electrophoresed and transferred to a nitrocellulose membrane, the membrane was blocked and incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: AdipoR1 (1:1000, ab126611), p-AMPKα (1:1000, ab133448), AMPKα (1:1000, ab32047), p-NFκB (1:1000, ab86299), NFκB (1:2000, ab16502), tumor necrosis factorα (TNFα) (1:1000, ab6671), interleukin-6 (IL-6) (1:1000, ab6672) (all from Abcam, MA), CTRP9 (1:500, NBP2–46834, Novus, CO), and adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) (1:1000, sc-271,901, Santa Cruz Biotechnology, CA). β-actin was used as an internal loading control. The respective secondary antibodies were incubated for 1 h at room temperature. The bands were probed with an ECL Plus chemiluminescence reagent Kit (Amersham Biosciences, Arlington Heights, PA) and visualized with the image system (Bio-Rad, Versa Doc, model 4000). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, NIH, USA).

Sections of the formalin‐fixed paraffin‐embedded brain tissue specimens were cut into 4 μm slides, dewaxed with xylene (twice, 15 minutes each time) and hydrated in an alcohol gradient (100%, 95%, 85% and 75%, 5 minutes in each concentration). Thereafter, the slides were stained with haematoxylin and eosin (H&E) to assess the degree of brain injury. The hydrated slides were processed for antigen retrieval under high temperature and pressure (3 minutes), and blocked with goat serum before immunofluorescence staining. Subsequently, the slides were incubated overnight at 4°C with primary antibodies against the following proteins: SESN2 (PA5‐72834, Thermo Fisher, MA, USA), NeuN (MAB377, EMD Millipore, Billerica, MA, USA), GFAP (MAB360, EMD Millipore Billerica, MA, USA), p‐AMPK (ab32047, Abcam, CA, UK), p‐mTOR (ab109268, Abcam, CA, UK) and p‐ULK1 (ab203207, Abcam, CA, UK). The sections were then incubated with either fluorescein isothiocyanate/tetramethylrhodamine‐conjugated antimouse, or rabbit IgG antibodies (Thermo Fisher, MA, USA), at 37°C for 1 hour. Finally, nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (Beyotime, Beijing, China), and a total of 6 sections per group were analysed under a DX51 microscope (Nikon Corporation, Tokyo, Japan).