Gpr124

At 72 h after ME, mice were anesthetized and transcardially perfused with PBS, followed by perfusion with cold 4% PFA (w/v) in PBS. Whole brains were immediately removed and fixed overnight at 4 ℃. The specimens were then cryoprotected with 30% sucrose in PBS and sectioned into the 45-μm-thick sample on a freezing microtome 31 (link). The sections were incubated with 0.1% Triton X-100 in PBS for 15 min at RT and for another hour in 3% BSA in PBS. The sections were incubated with primary antibodies, followed by staining with corresponding secondary antibodies. After staining with DAPI, sections were then washed with PBS and mounted onto glass slides with Vectashield mounting medium (Vector Laboratories). Images were acquired using a NikonA1R confocal microscope.
The following primary antibodies were used: rabbit anti-GPCR GPR124 (GPR124, 1:300, Abcam, ab67820), goat anti-CD13 (1:200, R&D systems, AF2335), Fluorescein lycopersicon esculentum (tomato) lectin (1:200, Vector Laboratories, FL-1171), Rabbit anti-Nitrotyrosine (1:1,000, Millipore, 06-284), rabbit anti-Ki67 (1:200, Abcam, ab66155).

Briefly, cells were fixed in 4% PFA as previously reported 31 (link). The cells were incubated with 0.1% Triton X-100 in PBS for 10 min at RT and for another hour in PBS containing 3% bovine serum albumin (BSA). The cells were incubated with primary antibodies, followed by labeling with corresponding fluorescent secondary antibodies. After staining with DAPI, cells were then rinsed with PBS and mounted onto glass slides with Vectashield mounting medium (Vector Laboratories). Images were captured using a NikonA1R confocal microscope.
The following primary antibodies were used: rabbit anti-GPCR GPR124 (GPR124, 1:300, Abcam, ab67820), rabbit anti-GPR124 (TEM5, 1:500, Invitrogen, PA5-20442), mouse anti-Vinculin (1:300, Abcam, ab18058), mouse anti-Paxillin (1:300, Thermofisher, MA5-13356), Alexa Fluor 647 Phalloidin (1:40, Invitrogen, A22287), Alexa Fluor 555 Phalloidin (1:40, Invitrogen, A34055), mouse anti-Myc-tag (1:2,000, Medical & biological laboratories, M192-3), goat anti-PDGFR-β (1:300, R&D systems, AF1042), rabbit anti-Partitioning-defective 3 (PAR-3, 1:500, Millipore, 07-330).
Isolated focal adhesions were stained with the following antibodies: rabbit anti- GPR124 (1:300, Abcam, ab67820), mouse anti-Vinculin (1:300, Abcam, ab18058), mouse anti-Paxillin (1:300, Thermofisher, MA5-13356).