The harvested samples were fixed with PFA for 30 min and then soaked overnight in 30% sucrose solution at 4°C on a nutating tube rocker. Next, they were immersed in optimal cutting temperature compound (23-730-571, Fisher Scientific) and placed in a cryostat set at −20°C. Cryosections of 40- or 60-μm thick were made and placed on poly-l -lysine (0.1%, w/v)–coated slides.
The cryosectioned samples were gently washed with DPBS, permeabilized with 0.1% Triton X-100 (T8787, Sigma-Aldrich), and blocked with 2% bovine serum albumin (A2153, Sigma-Aldrich). Primary rabbit VE-cadherin antibody (2158, Cell Signaling Technology) was diluted 1:200 in cell staining buffer (420201, BioLegend) and incubated with the samples at 4°C overnight. The primary antibody was then labeled with donkey anti-rabbit immunoglobulin G CF543 secondary antibody (20308-1, Biotium), which was diluted in cell staining buffer at 1:200 and incubated at 37°C for 2 hours. Cytoskeleton and nuclei were labeled with Phalloidin eFluor 660 (50-6559-05, eBioscience) and DAPI (4083S, Cell Signaling Technology) per the manufacturer’s instruction before the slides were mounted with antifade reagent (9071S, Cell Signaling Technology).
The cryosectioned samples were gently washed with DPBS, permeabilized with 0.1% Triton X-100 (T8787, Sigma-Aldrich), and blocked with 2% bovine serum albumin (A2153, Sigma-Aldrich). Primary rabbit VE-cadherin antibody (2158, Cell Signaling Technology) was diluted 1:200 in cell staining buffer (420201, BioLegend) and incubated with the samples at 4°C overnight. The primary antibody was then labeled with donkey anti-rabbit immunoglobulin G CF543 secondary antibody (20308-1, Biotium), which was diluted in cell staining buffer at 1:200 and incubated at 37°C for 2 hours. Cytoskeleton and nuclei were labeled with Phalloidin eFluor 660 (50-6559-05, eBioscience) and DAPI (4083S, Cell Signaling Technology) per the manufacturer’s instruction before the slides were mounted with antifade reagent (9071S, Cell Signaling Technology).