Anti cd86 bv510

DRG single-cell suspensions were blocked for non-specific antibody-binding with anti-CD16/32 Fc-Block (BioLegend 101320, 1:100) and incubated in an antibody cocktail consisting of anti-CD163-PE (BioLegend 156703, 1:200), anti-TLR4-PE/Cy7 (BioLegend 117609, 1:200), anti-CD206-PerCP/Cy5.5 (BioLegend 141715, 1:200), anti-CD11b-APCCy7 (BioLegend 101225, 1:200), anti-CD68-Alexa700 (BioLegend 137025, 1:200), anti-CD80-Bv421 (BioLegend 104725, 1:200), anti-CD86-Bv510 (BioLegend 105039, 1:200), anti-Gr1-Bv605 (BioLegend 10844, 1:200), anti-F4/80-Bv785 (BioLegend 123141, 1:200), and anti-CX3CR1-FITC (BioLegend 149019, 1:200) for 20 min at 4 °C. Exclusion of dead cells was achieved by resuspending cells in FACS buffer containing 10 nM TO-PRO-3 (Invitrogen, Carlsbad, CA, USA, T3605). Flow cytometry from the samples was performed using a BD LSRFortessa device and BD FACSDiva Software (BD Biosciences). Raw data were analyzed using the CytoExplorer R package version 1.1.0 [46 ].

For brain tissues, we homogenized the hemisphere ipsilateral to the infarct of tMCAO or sham mice using Neural Tissue Dissociation Kit (130–093-231, Miltenyi Biotec) by the gentle MACS Dissociator following the manufacturer’s instructions. The immune-cell‐enriched population was collected using Percoll gradient centrifugation. Isolated cells were stained with anti-CD11b-FITC (101,206, Biolegend), anti-CD86-BV510 (105,040, Biolegend), anti-CD45-APC-Cy7 (103,116, Biolegend), and anti-CD206-APC (141,708, Biolegend). Flow cytometry was performed on BD FACSVerse (BD Bioscience) and FlowJo software (TreeStar) was used to analyze the data.