Percp cytm5.5 anti mouse cd45 antibody

Animal subjects were sacrificed by an overdose of 2% pentobarbital sodium and their brains were carefully extracted and dissected. To dissociate the tissues, a digestion process using Papain (2 mg.ml^− 1, Worthington) in RPMI 1640 medium (Gibico) was carried out at 37℃ for 1 h. The resulting dispersed cells were then filtered using the 70 mm nylon mesh. Subsequently, the resulting cells were further resuspended with a 30% Percoll density gradient (GE Healthcare) and centrifuged at 900 g for 25 min. The cells were isolated by collecting the bottom fraction in the 30% Percoll solution. The samples were then treated with FcR Blocking Reagent (Miltenyi Biotec) for blocking and subjected to flow cytometry analysis for the detection of surface antigens. For fluorescence acquisition, the cells were stained with FITC anti-mouse CD11b Antibody (BioLegend) and PerCp-CyTM5.5 anti-mouse CD45 Antibody (BD Pharmingen). Flow cytometry (FACS Aria II SORP, BD Biosciences, USA) was employed for fluorescence acquisition, and the samples were gated based on CD11b + CD45dim expression, which represents the microglia population.

The peri-infarct tissues of mice were collected and then temporarily placed on ice. Tissues were dissociated and digested for 1 h at 37 °C by Papain (2 mg/mL, LS003119, Worthington) in RPMI 1640 medium (C11875500BT, Gibico). The mixture was passed through a 70-μm nylon mesh. Dispersed cells were collected by centrifugation with 300 × g for 10 min. The cell pellet was resuspended in 30% Percoll density gradient (17089109, Cytiva) and centrifuged at 900 × g for 25 min. Samples were blocked with FcR Blocking Reagent (130-092-575, Miltenyi Biotec) and resuspended in PBS containing 2% FBS. The different cells were assayed for surface antigens by flow cytometry as previously described^{17 (link)}. Cells were used for fluorescence acquisition and stained with APC anti-Mouse NCAM-1/CD56 Allophycocyanin MAb (FAB7820A-100, R&D), PE anti-mouse ACSA-2 (130-116-244, Miltenyi Biotec), FITC anti-mouse/human CD11b Antibody (101205, BioLegend), PerCp-CyTM5.5 anti-mouse CD45 Antibody (561869, BD Pharmingen), and Brilliant Violet 605™ anti-mouse CD31 (102427, BioLegend). Gating was determined based on the appropriate negative isotype-stained controls. Flow cytometry (BD Biosciences, FACSAria II SORP) was used for fluorescence acquisition, and data were analyzed using FlowJo-V10 software.