Anti mouse cd31 alexa fluor 488

To isolate adult endothelial cells from mouse kidney, liver, heart, and lung, mice were injected intravitally with 25 μg of anti-VE-cadherin-AF647 antibody (clone BV13, Biolegend) retro-orbitally in 6–8-week-old male C57BL/6J mice under anesthesia 10 min before they were killed and the organs harvested. For cell sorting, organs were minced and incubated with Collagenase A (25 mg ml⁻¹) and Dispase II (25 mg ml⁻¹) at 37 °C for 20–30 min to create a single-cell suspension. Cells were filtered through a 40-μm filter immediately before counter staining. The single-cell suspension was first blocked with an Fc-quenching antibody before antibody staining with anti-mouse CD31-Alexa Fluor® 488 (102414, Biolegend), anti-mouse CD45-Pacific Blue™ (103126, Biolegend), and anti-mouse Podoplanin-PE/Cy7(127412, Biolegend). Embryonic tissues were dissected and processed through the same antibodies. Following staining, cells were processed for FACs sorting.

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).