Ab150078 ab150114

HUVECs on glasses were fixed with 4% paraformaldehyde for 20 min, permeabilized with cold phosphate buffered saline (PBS) containing 0.01% Triton X-100 for 10 min, incubated with blocking buffer (3% bovine serum albumin in PBS) for 60 min, and then incubated overnight with primary antibodies against ZO1 (1:50), Occludin (1:30), β-catenin (1:100), Nesprin1 (1:100, Huabio ET7107-28), SUN2 (1:100, Millipore MABT880). The cells were washed with PBS, and incubated with secondary antibody, such as Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (1:500, Abcam ab150077/150113) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (1:500, Abcam ab150078/ab150114). The nuclei were stained with DAPI. The mounted slides or slips were visualized by fluorescence microscopy (Leica DMI6000B or Leica TCS SP8).

Tissues were first washed with PBS buffer and adventitia was removed carefully, and then they were fixed with 4% paraformaldehyde (PFA) and embedded in 20% sucrose solution before being frozen in TissueTek cutting medium (Sakura Finetek). 8 μm sections were processed for immunofluorescent analysis. The sections were further fixed with 4% PFA for 20 minutes. For immunostaining of attached cells, cells were fixed in 4% PFA for 20 minutes and permeabilized with 0.1% Triton X-100 (in PBS) for 5 minutes and rinsed for 3 times. Nonspecific binding was blocked by 4% BSA in PBS for 1 hour. Tissues/cells were incubated at 4 °C overnight in incubation buffer containing 4% BSA and the primary antibodies including anti-CDH5 (1:100), anti-Ki67 (Abcam, ab15580, 1:200), anti-F4/80 (BioLegend, 157309, 1:100), anti-SM22α (Proteintech, 10493-1-AP, 1:100), anti-ALDH2 (1:100), anti-ALDH3A1(1:100), anti-ALDH6A1 (1:100), and anti-pSmad2/3 (1:100). After being washed in PBS for 3 times, the specimens were incubated with Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150077/ab150113) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150078/ab150114) or Alexa Fluor® 647 goat anti-rabbit igG (Abcam, ab150083) for 1 hour at room temperature. The fluorescent signals were detected by fluorescence microscopy (Leica TCS SP8).

For immunofluorescence, cells were fixed in 4% PFA for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 to 10 min. Nonspecific binding was blocked by 3% BSA for 30 min. The primary antibody was diluted 1:200 in 3% BSA and incubated overnight at 4 °C, then probed with secondary antibody including Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (ab150077/ab150113; Abcam) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (ab150078/ab150114; Abcam) diluted 1:200 in PBS for 1 h at room temperature. Nuclei were counterstained with DAPI. Images in either fixed cells or live cells were captured by using Leica SP8 confocal microscope or stimulated emission depletion (STED) super-resolution microscopy.
For quantification of subcellular distribution, the regions of cell nucleus or cytoplasm were manually determined, and measurements of fluorescence intensity were conducted by using NIH ImageJ software. Statistic data were obtained from approximate 100 ~ 200 cells from 18 random fields from 3 independent experiments. Each dot represents the mean value of all the cells from one random field.