Dylight 488 green

Kidney cryosections were mounted on polylysine-coated slides after immersion and fixed in ice-cold acetone for 10 min, washed three times in PBS, permeabilized in 0.1% Triton X-100 in PBS for 10 min, blocked in 0.5% BSA, and incubated with primary antibodies at 4 °C overnight. After washing, sections were incubated for 1 h at room temperature with the appropriate DyLight 488 (green) or DyLight 594 (red)-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories. DAPI was used to stain the nuclei, and images were captured using a confocal microscope. Immunofluorescence labeling was performed on P-MCs in culture after they had been fixed with 4% paraformaldehyde (wt/vol) (PFA) for 15 min on a rocking platform at room temperature.

A standard IF staining protocol was conducted, respectively, in live and fixed cells to measure the Palladin expression. Primary antibody against Palladin (Cat No. MA5‐16141, Invitrogen) and secondary antibody prelabeled with DyLight 488 (green, Cat No. 711–486‐152, Jackson Immunoresearch) were used in this study. The nuclear were stained with 4,6‐diamidino‐2‐phenylindole (DAPI). The fluorescent images were acquired through fluorescence microscopy (Nikon).