Anti mouse cd45 apc

For the flow cytometry analysis of the peripheral blood cell detection, we used the strategy recommended by Liu et al. [18 (link)]. The samples were preincubated with anti-mouse CD16/CD32 (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-092-574) for 15 min. Then the following antibodies (all from Miltenyi Biotec) were used: anti-mouse Ly6G–VioBlue (130-119-902), anti-mouse FcεR1–PE (130-118-896), anti-mouse SiglecF–PE-Vio615 (130-112-330), anti-mouse CD172–PE-Vio770 (130-123-154), anti-mouse CD45–APC (130-110-798), anti-mouse CD11b–APC-Vio770 (130-113-803). The antibodies were used in dilution 1:30. The samples were incubated for 30 min and washed twice with DPBS. SytoxGreen (Invitrogen, Eugene, OR, USA, S34859) was added (in dilution 1:1,000,000) 5 min before the acquisition. The measurement was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec).

Control (C) and chronically stressed mice (CS) were perfused transcardially with 0.9% saline. Brains were quickly removed. The ipsilateral ischemic MCA territory, as well as the corresponding area in the contralateral hemisphere, was carefully dissected and placed in cold PBS (4 °C). Brain tissue of 3–5 animals was pooled. CD31+ cells were enriched from brain tissue using a MACS protocol combined with prior myelin removal (Miltenyi Biotec GmbH). To ensure a high purity of ECs, an additional FACS step was performed. Cells were stained with DAPI and antibodies against CD31, CD146, and CD45. Single cells that were CD45-/DAPI-/CD31+/CD146+ were separated from the remainder of the suspension using a BD FACSAria™ II flow cytometer. For FACS staining, the following antibodies were used: anti-mouse CD31-Alexa Fluor® 488 (#102514, BioLegend, Inc.); anti-mouse CD45-APC (#130-102-544, Miltenyi Biotec GmbH); anti-mouse CD146 (LSEC)-PE (#130-102-319, Miltenyi Biotec GmbH).