Apc conjugated cd31 antibody

Fixed kidney organoids were incubated in blocking buffer (PBS 1X donkey serum 10% triton X100 0.3%) at 4°C for 3h before adding primary antibodies against HNF4α (Life Technologies 1:300, cat# MA1-199), Nephrin (NPHS1 1:300, Bioscientific, cat# AF4269) and Claudin-1 (CLDN1 1:100, Thermo Fisher Scientific, cat# 71-7800) at 4°C for 2 days. After washing in PBS 1X triton X-100 0.1%, organoids were incubated in secondary antibodies 1:400 at 4°C for 2 days: Alexa fluor 405 donkey anti-mouse (Abcam, cat# ab175659), Alexa fluor 488 donkey anti-goat (Molecular Probes, cat# A11055) and Alexa fluor 568 donkey anti-rabbit (Life Technologies, cat# A10042). Samples were then washed before blocking at 4°C for 3h with PBS 1X mouse serum 10μg/ml triton X-100 0.3% and adding an APC-conjugated CD31 antibody (1:50, Biolegend, cat# 303115) at 4°C for 2 days. Finally, samples were washed and imaged in 50:50 glycerol:PBS 1X using a Dragonfly Spinning Disc Confocal Microscope (Andor Technology).

After removal of the surrounding connective tissue, femurs were dissected from the three groups of mice. Femurs were clipped in DPBS digested with 2.5 mg/mL collagenase A (Biosharp, Hefei, China) and enzymatically with 1 unit/mL dispersible enzyme I (Biosharp, Hefei, China) and kept under gentle agitation at 37°C for 15 min. The obtained cell suspension was filtered using a 40-um cell strainer (Biosharp, Hefei, China) and washed with DPBS. After washing, the cells were incubated with FITC-conjugated EMCN antibody (Santa Cruz Biotechnology, USA) and APC-conjugated CD31 antibody (BioLegend, San Diego, CA) for 30 min at room temperature. Thereafter, the cells were resuspended in DPBS. All samples were analyzed using the CytoFLEX LX Flow Cytometry System (CytoFLEX, Backman Counter, CA, USA) and CytExpert 2.4 Software.