Pa5 87933

Total proteins were extracted using tissue protein extraction reagents. Total tissue proteins (20 µg/lane) were loaded and separated on sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE) at 120 V for 90 min, followed by transferring to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) at 300 mA for 2 h. After that, Membranes were incubated in 5% (w/v) milk for 1 h followed by incubation overnight at 4 °C with primary antibodies, including, rabbit anti-TREM2 (1:1000, PAB37053, Bioswamp, China), rabbit anti-TREM2 (1:1000, PA5-87933, ThermoFisher, USA), rabbit anti-MBP (1:1000, 78896S, Cell Signaling Technology, USA), Acvr2B (1:1000, ab128544 and ab180185, Abcam, UK), mouse anti-GAPDH (1:1000, T0004, Affinity, USA), rabbit anti-β-actin (1:1000, AF7018, Affinity, USA) overnight at 4 °C. After washes, the membranes were incubated with corresponding secondary antibodies for 1 h. The ECL western blotting detection kit (P007, ABP Biosciences, Japan) and an enhanced chemiluminescence system were used to examine protein bands. ImageJ software was used to analyze the gray value of protein bands.

For immunofluorescence staining, brain sections were pretreated with citrate buffer for 5 min for antigen retrieval, then blocked with immunostaining blocking solution (P0102, Beyotime, China) at room temperature for 1 h. Then the sections were incubated with rabbit anti-Oligo2 (1:100, ab109186, Abcam, UK) overnight at 4 °C. For double immunofluorescence staining, the sections were incubated with mixtures of rabbit anti-IBA-1 (1:300, 019-19741, Wako, Japan) or mouse anti-IBA-1 (1:300, 016-26721, Wako, Japan) and rabbit anti-TMEM119 (1:100, NBP2-30551, Novus, USA), mouse anti-iNOS (1:100, ab210823, Abcam, UK), rabbit anti-CD206 (1:100, ab64693, Abcam, UK), rabbit anti-TREM2 (1:200, PA5-87933, ThermoFisher, USA), and mouse anti-activin-A (1:50, ab89307, Abcam, UK); and mixtures of rabbit anti-NG2 (1:100, AB5320, Millipore, USA) and mouse anti-activin-A (1:50, ab89307, Abcam, UK), Acvr2B (1:100, ab128544 and ab180185, Abcam, UK); The next day, sections were incubated with secondary antibodies after washing with phosphate-buffered solution (PBS). In addition, sections were incubated with Rabbit anti-BCAS1 FITC conjugated (1:50, C01226F, SAB, China). Finally, sections were mounted with a solution containing 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were observed under a microscope (BX63, Olympus).