Cy3 secondary antibodies

hASCs were seeded on coverslips in 12-well plates to detect the effect of osteoinduction on polycystin-2 and the effect of PC2 knockdown on primary cilia formation by immunofluorescence. After 48 h, hASCs were fixed with 4% paraformal dehyde for 15 min and washed three times with PBS. Triton X-100 (0.5%, Sigma-Aldrich, United States ) was added to each well for 20 min at room temperature. After washing three times with PBS, hASCs were blocked with goat serum for 1 h. Primary antibodies specific for polycystin-2 (1:50, SANTA CRUZ, United States ) and acetylated α-tubulin antibody (diluted 1:100) (Sigma, Catalog #T7451) were added to the cells and then incubated overnight at 4°C. After washing three times with PBS Tween-20, fluorescent Cy3 secondary antibodies (1:50, Proteintech, United States ) were added and incubated for 1 h at 37 °C in the dark. The nucleus was then restained with DAPI (Sigma-Aldrich, United States ). Cells were subsequently viewed by fluorescence microscopy (ZEISS, Oberkochen, Germany).

After transfecting process mentioned in transfecting of tools for nucleic acid expression, the effect of miR-503-3p, Wnt2, and Wnt7b on the expression of β-catenin protein and their location were detected by immunofluorescence. After loading cyclic strain, samples were fixed by 4% paraformaldehyde in wells of BioFlex™ plates, then rinsing 3 times by PBS. At room temperature, 0.5% Triton X-100 (Sigma-Aldrich, USA) was added for transpiring them for 20 min. Goat serum (Beyotime, China) was used to block hASCs for 2 h and then incubated with primary antibodies specific for β-catenin (1:1000, Abcam, USA) overnight at 4 °C. After it is rinsed thrice with PBS with Tween-20, cells were incubated with fluorescent Cy3 secondary antibodies (1:50, Proteintech, USA) for 1 h at 37 °C in the dark. Sample photos were taken by fluorescence microscopy (ZEISS, Germany).

Cells were plated onto coverslips and fixed in 4% paraformaldehyde for 20 min when cells grew to 50% confluency. Then, the coverslips were permeabilized with or without 0.3% Triton, incubated with goat serum for 30 min and primary antibody overnight at 4 ℃. After it was rinsed thrice with PBS with Tween-20, cells were.
incubated with fluorescent Cy3 secondary antibodies (1:50, Proteintech, USA) for 1 h at 37 °C in the dark. After the slides were incubated with DAPI (Life Technologies, USA), cells were observed and photographed under an FV1000 laser confocal scanning microscope (Tokyo, Japan). Primary antibodies used were as follows: E-cadherin (1:100, CST), Vimentin (1:100, CST), and β-catenin (1:100; CST).