Ab70362

OCCM-30 cells were cultured overnight on sterile Falcon™ Chambered Cell Culture Slides (354108, Fisher Scientific) and further fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% Triton™ X-100 Surfact-Amps™ Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were incubated in blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma) and 0.1% Tween-20 (P1379, Sigma-Aldrich) for 30 min at room temperature and further incubated with primary antibodies AdipoR1 (ab70362, Abcam) (dilution 1:250) or AdipoR2 (ab77612, Abcam) (dilution 1:250) at 4°C overnight. The secondary antibodies DyLight 488 polyclonal goat anti-rabbit (ab96899, Abcam) (dilution 1:500) or donkey anti-goat Alexa Fluor 647 (ab150131, Abcam) (dilution 1:500) conjugated to fluorescein isothiocyanate were used. After washing with 1× phosphate-buffered saline (PBS) (10010023, Thermo-Fisher), samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.

Double Immunofluorescence staining was conducted on the sections of fixed frozen brains as previously described (19 (link), 20 (link)). The 8-μm thick slides were rinsed using phosphate-buffered saline (PBS), followed by permeabilization using 0.3% Triton X-100 for 15 min at RT. Next, the slides were incubated with blocking solution (95% PBS, 5% normal donkey serum, and 0.05% Triton X-100) for 2 h. Next, the slides were incubated using the following: mouse anti-Iba1 (ab283319, 1:200, Abcam, USA), rabbit anti-GFAP (ab254082, 1:50, Abcam, USA), rabbit anti-APN (ab181281, 1:50, Abcam, USA), rabbit anti-AdipoR1 (ab70362, 1:100, Abcam, USA), mouse anti-NeuN (ab104224, 1:200, Abcam, USA), rabbit anti-IL-6 (ab179570, 1:100, Abcam, USA), rabbit anti-CD68 (ab283654, 1:100, Abcam, USA), anti-hemoglobin (ab251919, 1:100, Abcam, USA) or rabbit anti-CD206 (ab64693, 1:100, Abcam, USA) at 4°C overnight. After using PBS to wash the slides three times (10 min each time), the sections were incubated using the appropriate fluorescent secondary antibodies (diluted 1:200) (Jackson Immuno Research, West Grove, PA, USA) and counterstained with DAPI (SKU: H-1200-10, Vector Laboratories, Newark, CA, USA). All counts and quantifications were conducted blindly (19 (link), 21 (link)).

Antibodies against to AMPKα (#5831), phospho-AMPKα (Thr172) (#2535), p38 MAPK (#8690), phospho-p38 MAPK (Thr180/Tyr182) (#9216), caspase-3 (#9662), cleaved caspase-3 (#9661), Myc-tag (#2276), ubiquitin (#3936), β-tubulin (#2146) were from Cell Signaling Technology (Billerica, MA, USA). Antibodies against to AdipoR1 (ab70362), AdipoR2 (ab77612), and HSP60 (ab46798) were obtained from Abcam (Cambridge, MA, USA). Normal IgG (sc-2025) and secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abbiotec (San Diego, CA, USA), respectively. Recombinant mouse adiponectin (ALX-522-059) and recombinant rat adiponectin globular form (Catalog#: SRP4593) were acquired from Enzo Life Sciences (Farmingdale, NY, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. MG132 (HY-13259) was obtained from MedChemExpress (Monmouth Junction, NJ, USA).