Rabbit anti ve cadherin

After 14 days in differentiation medium, immunofluorescence staining was performed on induced and un-induced cells to detect the expression of CD31, VE-Cadherin and vWF. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS twice, blocked with 1% bovine serum albumin (BSA) for 1 h at 37° C, and incubated with AlexaFluor-488-conjugated Mouse anti-CD31 (Abcam), Rabbit anti-VE Cadherin (Abcam) and Rabbit vWF (Abcam) antibodies at 4° C overnight, followed by washing in PBS three times. AlexaFluor-555-conjugated goat anti-rabbit secondary antibody (ThemoFisher Scientific) was then added to VE-Cadherin and vWF stained samples and incubated for a further 1 h. Cells were then counterstained with DAPI (Sigma) and observed under a confocal microscope.

For in situ immunofluorescence of hRVTU, devices were cellularized, cultured for the intended duration, and fixed via perfusion of 3.7% paraformaldehyde through the intact device for 20 min followed by washing with PBS three times. For immunofluorescence of isolated fetal kidney cells, P3 cells were cultured on top of glass coverslips for 8 days and then fixed with 3.7% paraformaldehyde. Both hRVTU and coverslips were incubated for one hour in 2% BSA and 0.1% Triton-X in PBS for blocking and membrane permeabilization. Primary antibody staining was performed overnight at 4°C. Primary antibodies and dilutions used were: rabbit anti-VE-cadherin (1:50-1:100 dilutions, Abcam), mouse anti-E-cadherin (1:50, Abcam), Phalloidin-568 primary-conjugated antibody (1:50 dilution, Invitrogen), FITC-conjugated sheep anti-VWF (1:100, Abcam), mouse anti-acetylated tubulin (1:10000 dilution, Sigma-Aldrich), rabbit anti-SGLT2 (1:50 dilution, Abcam), and rabbit anti-ADP-ribosylation factor-like protein 13B (1:10000, Proteintech). Imaging was performed on a Nikon A1R confocal microscope with image analysis conducted in ImageJ software^{[49 (link)]} (U.S. National Institute of Health, Bethesda, Maryland) and three-dimensional rendering performed using Imaris software (Bitplane).