Goat anti cd13

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).

Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and perfused transcardially with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). Brain tissue was collected and post-fixed for 6 h in 4% PFA. For cryoprotection, tissue was transferred to 30% sucrose and stored at 4°C. Coronal sections were cut at a thickness of 40 µm using a sliding microtome (Microm HM430, Leica), collected, and stored as free-floating sections in cryoprotectant solution at −20°C.
For immunostaining, brain sections were blocked with 5% normal donkey serum for 1 h at room temperature and incubated with primary antibodies (rabbit anti-GFAP 1:200, Dako, #GA524; goat anti-Iba1, 1:500 Wako, #011-27991; NeuroTrace™ 1:200, Thermo Fischer; mouse anti-NeuN Antibody 1:500, Merck, #MAB377; rabbit anti-Neurofilament 200 antibody 1:200, Merck, #N4142; guinea pig anti-Neurofilament L, 1:200, Synaptic Systems, rat anti-CD31 antibody 1:50, BD Biosciences, #MEC13.3; goat anti-CD13, 1:200; R&D Systems, #AF2335) overnight at 4°C. The next day, sections were incubated with corresponding secondary antibodies (1:500, Thermo Fischer Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Mounting was performed using Mowiol.

For immunohistochemistry, brain sections were washed with 0.1 M phosphate buffer (PB) and then incubated for 30 minutes at room temperature in a blocking solution containing TNB, TBST (0.1%) and normal goat serum (3%). For detection of vascular endothelial cells, sections were incubated overnight at 4 °C with monoclonal rat anti-CD31 antibody (BD Biosciences, 1:50). Tight junction proteins were detected with the following antibodies: mouse anti-Claudin-5 antibody (1:200, ThermoFischer); rat anti-VE-Cadherin antibody (1:100, ThermoFischer) and rabbit anti-ZO-1 antibody (1:100, ThermoFischer). Pericytes were visualized with goat anti-CD13 (1:200; R&D). The primary antibody incubation was followed by 2 h incubation at room temperature with corresponding fluorescence secondary antibodies (1:500, ThermoFisher). DAPI (1:2000 in 0.1 M PB, Sigma) was used to visualize nuclei. Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides and coverslipped using Mowiol.