4 6 diamino 2 phenylindole dapi

XR cells were incubated with 5 µmol/L 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Dojindo, Kumamoto, Japan) for 30 min at 37 °C. BCECF-AM was then converted to BCECF in the cytoplasm, and BCECF-loaded XR cells were washed twice with PBS prior to being used for in vivo experiments. The cells were diluted to the appropriate concentration with PBS, and used immediately for experiments. At 48 h post-MCAO, 1 × 10⁵ BCECF-loaded XR cells in 50 µl PBS/mouse were injected into the carotid artery. At 10 min, 30 min, 1 h, and 3 h post-cell transplantation, the mice were euthanized and then decapitated (n = 3). After removal from the skull, the brains were sectioned coronally (20 µm) using a vibratome (Leica, Wetzlar, Germany), and stained with antibodies against CD31 (BD Biosciences, NJ USA) and glial fibrillary acidic protein (GFAP; Thermo Fisher Scientific, MA, USA). Primary antibodies were visualized using Alexa Fluor 555- or 647-conjugated secondary antibodies (Molecular Probes, OR, USA), and the cell nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI; Kirkegaard & Perry Laboratories, MD, USA).