Pe rat anti mouse cd144

Bone marrow endothelial cells (ECs) were stained by intravenous injection of 10 μg anti-VE-cadherin antibody (BD Biosciences, PE Rat Anti-Mouse CD144, #562243) 5-10 min before euthanizing the mice. Tibias and femurs were gently crushed using a mortar and pestle and then digested with DNase I (200 U/mL; ThermoFisher, # EN0521), Liberase^DL (250 mg/mL; Sigma, # 5401160001), type IV collagenase (1 mg/mL; ThermoFisher, # 17104019), and collagenase D (500 μg/mL; Sigma, # 11088858001) with agitation for 30 min at 37 °C. The cells were dissociated to a single-cell suspension by passing through a 25G needle several times and filtered with a 70-µm nylon mesh to generate a single-cell suspension. Cells were sorted in two successive rounds to ensure high purity using a FACS Aria II flow cytometer.

5 × 10⁴ ECs were cultured in 24-well plates for 24 h and fixed with 10% formalin for 10 min and blocked with blocking buffer containing 5% bovine serum albumin (BSA, bioWORLD) in 1X DPBS to block non-specific binding sites for 1 h at RT. The cells were then stained with either 0.01 mg/ml PE Rat Anti-Mouse CD31 (553373, BD Biosciences) or 0.01 mg/ml PE Rat Anti-Mouse CD144 (562243, BD Biosciences) antibodies in a staining buffer containing 1% BSA in 1X DPBS and incubated at 4°C overnight. The cell nuclei were stained with 1:5000 DAPI for 5 min and washed 3 times. Then images were taken using a Zeiss Observer Z1 microscope.