Goat serum

The paraffin-embedded tissue sections were hydrated in xylene (Guangcheng Chemical Reagent Co., Ltd., Tianjin, China) and a graded alcohol series. Antigen retrieval was performed in a water bath at 95°C for 20 min with citric acid buffer (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and endogenous peroxidase activity was blocked with 3% H₂O₂. Next, the tissue sections were incubated with goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 45 min and stained with rabbit anti-BMP-2 polyclonal antibody (cat. no. SAB1411278; 1:200 dilution; Sigma-Aldrich, Shanghai, China) and mouse anti-nNOS monoclonal antibody (cat. no. sc-5302; 1:200 dilution Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Following washing with PBS, the tissues were respectively incubated with the biotin-labeled goat anti-rabbit IgG secondary antibody (cat. no. ZDR-5306; 1:800 dilution; Zhongshan Golden Bridge Biological Technology Inc.) and the biotin-labeled goat anti-mouse IgG secondary antibody for 30 min at 37°C (cat. no. ZDR-5307; 1:800 dilution; Zhong Shan Golden Bridge Biological Technology Inc, Beijing, China). Then tissues were stained with 3,3′-diaminobenzidine and hematoxylin (Zhongshan Golden Bridge Biological Technology Inc, Beijing, China). The experiment was repeated three times.

The cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) containing 1% phenylmethylsulfonyl fluoride. The BCA method was used to determine protein concentrations. An equal amount (25 µg) of each sample was separated by 6–10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% goat serum at 37°C for 30 min (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing China) and incubating with primary antibodies against p-gp (dilution, 1:300; cat. no. sc71557), MRP1 (dilution, 1:300; cat. no. sc7773), LRP (dilution, 1:300; cat. no. sc135975) and GAPDH (dilution, 1:1,000; cat. no. sc47724) overnight at 4°C, the PVDF membranes were washed three times with PBS and Tween-20 (PBST) buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (dilution, 1:5,000; cat. nos. TA130003 and TA130023) for 1 h at 37°C. The membranes were then washed three times with PBST buffer and the amount of protein in each band was observed using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology) and quantified using Quantity One 4.6 computer software. GAPDH was used as a loading control. The experiment was repeated five times.

BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells (1 × 10⁴cells/well) on coverslips were pre‐treated with vehicle or 10 μM LY294002 for 1 h and then treated with vehicle or 1 μg/mL of LPS for 24 h. Subsequently, the individual wells of cells were exposed to 0.4 µL fluorescence labeled latex beads (F8775, Invitrogen) at 37°C, 5% CO₂ for 1 h. After being washed, the cells were fixed in 4% paraformaldehyde and treated with 5% goat serum (Zhongshan Golden Bridge Biotechnology, Beijing, China). The cells were incubated with rabbit anti‐mouse Iba‐1 overnight at 4°C and stained with Alexa Fluor 488‐goat anti‐rabbit IgG (H + L) (Proteintech, CHI, USA), followed by counterstaining with DAPI. The fluorescent signals were captured under a fluorescent microscope (OLYMPUS) and further analyzed using Image J software (National Institutes of Health, Bethesda, USA). The percentages of positive phagocytic cells were determined by calculating the number of cells with fluorescence labeled latex beads (red signal) relative to total numbers of microglia (green signal).