Ab125016

Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors (P0013B, Beyotime Biotechnology), and the concentrations of total cellular protein were measured using a BCA assay kit (P0010, Beyotime Biotechnology). Total protein samples (40 μg/gel) were separated via 12% SDS/PAGE gel and transferred to PVDF membranes (IPVH00010, Merck Millipore, Germany) by electroblotting. After blocking in TBST with 5% skimmed milk powder for 1 hr at room temperature (RT), the membranes were incubated with primary antibodies, including anti-Fyn (ab125016, 1 : 1000, Abcam, Cambridge, MA, USA), anti-HLA-G (79769, 1 : 1000, Cell Signaling Technology, Danvers, CO, USA), anti-ERK1/2 (ab54230, 1 : 1000, Abcam), anti-p-ERK1/2 (ab201015, 1 : 1000, Abcam), anti-STAT3 (ab68153, 1 : 1500, Abcam), anti-p-STAT3 (9145, 1 : 2000, Cell Signaling Technology), and anti-GAPDH (AB-P-R 001, 1 : 1000, Goodhere Biotechnology, Hangzhou, China), overnight at 4°C. Then, the membranes were washed and incubated with HRP-labelled goat anti-rabbit secondary antibody (BOSTER Biotechnology, Wuhan, China) for 1 hr at RT. The blot signals were detected by the Odyssey infrared imaging system (LI-COR Biosciences) and analysed by Odyssey software (LI-COR Biosciences).

The mice were perfused with PBS (G4202, Servicebio, Wuhan, China) and 4% paraformaldehyde (30525‐89‐4, Nanjing Chemical Reagent, Nanjing, China). After dehydration, permeation and embeddedness, the brain tissues were sliced by 10 μM thickness and placed in the same position of the six glass slides, and three brain slides are pasted on each glass slide. Next, dewaxing and hydration were carried out. The slices were blocked in 10% goat serum / PBST at room temperature and incubated with primary antibodies overnight at 4℃. The dilutions of primary antibodies were used in the experiments included as follows: NeuN (1:500, ab177487, Abcam, Cambridge, MA, USA), Iba1 (1:500, NB100‐1028, Novus, Centennial, Colorado, USA), CD45 (1:200, ab40763, Abcam, Cambridge, MA, USA), Ly6G (1:200, #31469, CST, Boston, USA), IB4 (1:200, I21414, Invitrogen, Carlsbad, CA, USA), NF‐κB (p65) (1:200, ab32536, Abcam, Cambridge, MA, USA), Nrf‐2 (1:200, 16396–1‐AP, Proteintech, Chicago, USA) and Fyn (1:300, ab125016, Abcam, Cambridge, MA, USA). The slices were washed by PBST and incubated with secondary antibodies (1:1000 dilution, Invitrogen, Carlsbad, CA, USA) for 1h at room temperature. DAPI (C3619‐PI19B, Southern Biotech, Birmingham, AL, USA) was incubated for 10 minutes. The quantification of images was analysed with Image J software as previously described.