Ab36900

Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with 1% protease inhibitor (Sigma-Aldrich, Saint Louis, MO, USA). Protein concentration was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). Total protein (30-40 μg per sample) was resolved by SDS-PAGE using a 10% gel electrophoresis. Thereafter, protein content was transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) and then incubated with 5% nonfat milk in Tris-buffer saline containing 0.1% Tween 20 (TBST) for 1-2 hrs at room temperature. Next, the membrane was incubated overnight at 4°C with primary antibody against C1QTNF6 (ab36900, 1 : 1000 dilution, Abcam, USA) or GAPDH (AB0037, 1 : 5000 dilution, Abways, China). After washing with TBST, the membrane was then incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (#7074, Cell Signaling Technology, USA). Respective protein signals were visualized by enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). GAPDH was used as a loading control.

Mouse brain tissues were deparaffinized and hydrated, and then the antigen was extracted with sodium citrate buffer. Next, these tissues were blocked with goat serum for 1 h at room temperature. Then, these tissues were incubated with the antibodies at 4°C overnight. The antibodies used in this research were NeuN (Abcam, ab177487) and CTRP6 (Abcam, ab36900). In the second day, these tissues were incubated with Alexa Fluor 647 (red for CTRP6, cell signaling technology, USA) and Alexa Fluor 488 (green for NeuN, cell signaling technology, USA) for 2 hours. DAPI (Thermo Fisher Scientific, USA) was used for the staining the cell nuclei. Finally, these tissues were observed with the laser scanning confocal microscope (Olympus, Japan). These images were analyzed with Image J (National Institutes of Health, USA). The proportion of positive cells in Sev group, Sev+Ad-NC group and Sev+Ad-CTRP6 group were normalized to the Sham group.

The ipsilateral cerebral hemisphere, rat leukocytes, and cultured cells were homogenized in a lysis buffer containing phosphatase and protease inhibitors. The venous blood samples were obtained 24 h after MCAO surgery, and the leukocytes were separated as previously described. We used the following primary antibodies: anti‐interleukin 1 beta (IL‐1β) (Catalogue, AF‐501‐NA), anti‐tumor necrosis factor‐alpha (TNF‐α) (Abcam, ab205587), anti‐matrix metallopeptidase 9 (MMP‐9) (Abcam, ab76003), anti‐zonula occludens‐1 (ZO‐1) (Arigo, ARG55738), anti‐occludin (Abcam, ab216327), anti‐caspase‐3 (Abcam, ab13847; Affinity Biosciences, AF7022), anti‐C1QTNF6 (Abcam, ab36900), and anti‐β‐actin (Abcam, ab20272). Proteins were detected by incubation with horseradish peroxidase‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 60 min at room temperature, using an enhanced chemiluminescence kit (Millipore). Gray values of the protein bands were analyzed using AlphaEase FC software (Alpha Innotech).