After fixation, the tissue sections were sealed by 0.3% Triton X-100 in PBS and 5% goat serum protein (Beyotime, Shanghai, China) for 1 h, reacted with isolectin (1 : 1000, Santa Cruz Biotechnology) at 4°C overnight, and incubated further with secondary antibody labeled with Alexa Fluor 488 or Alexa Fluor 594 (1 : 1000, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 45 min. The images were captured with the help of a fluorescence microscope (Olympus, Japan) and a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany). Quantification of the CNV area was performed with the help of an image analysis software.
Cells were washed with PBS, fixed with 4% formaldehyde for 15 min, treated with 0.2% Triton X-100 PBS at 4°C for 15 min, sealed with 1% BSA for 1 h, and incubated with anti-SOX9 antibody (sc-166505, 1 : 100, Santa Cruz Biotechnology). After PBS washing for 3 times, the cells were incubated with Alexa Fluor 568-labled secondary antibody (1 : 200) for 30 min, stained with DAPI, fixed, and observed under an inverted fluorescence microscope.
Cells were washed with PBS, fixed with 4% formaldehyde for 15 min, treated with 0.2% Triton X-100 PBS at 4°C for 15 min, sealed with 1% BSA for 1 h, and incubated with anti-SOX9 antibody (sc-166505, 1 : 100, Santa Cruz Biotechnology). After PBS washing for 3 times, the cells were incubated with Alexa Fluor 568-labled secondary antibody (1 : 200) for 30 min, stained with DAPI, fixed, and observed under an inverted fluorescence microscope.