Anti ve cadherin primary antibody

HUVECs were grown to confluence on 8-well cell culture slides (Cat#30108, SPL). The cells were pre-treated with VEGF (2.5 nM) for an hour and treated with ginsenosides (F1 or Rh1, 10 μM) for another hour. Otherwise, the cells were co-treated with Csn-B (1 μM) and ginsenosides (F1 or Rh1, 10 μM) for an hour. After the treatment, the cells were washed with PBS and were fixed in 4% paraformaldehyde (Cat#P2031, Biosesang) for 20 min at room temperature. The slides were blocked in 1% bovine serum albumin (BSA) diluted in PBST (0.1% Tween 20 in PBS) for 30 min at room temperature. Then, they were incubated with anti-VE-cadherin primary antibody (1:50 dilution; Cat#sc-9989, Santa Cruz) at 4 °C overnight followed by FITC-conjugated secondary antibody (1:50 dilution; Cat#sc-516140, Santa Cruz). Nuclei were counterstained with 0.5 μg/ml of DAPI (Cat#D9542, Sigma Aldrich) and the cells were mounted with permanent aqueous mounting medium (Cat#M01, Biomeda). Immunofluorescence was visualized by confocal microscopy (LSM 780, Zeiss).

To observe any changes of VE-cadherin expression in the lung, rat lungs were collected gently and then fixed in 4% paraformaldehyde at 4°C. 24 hours later, lung tissues were embedded with optimal cutting temperature medium (OCT, Sakura Finetek USA, Inc., Torrance, CA, USA) and then frozen in −80°C. 24 hours later, lung tissues were cut into 5 μm thick sections. VE-cadherin was stained with anti-VE-cadherin primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and followed by a second antibody Fluorescein- (FITC-) AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Inc., PA, USA). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Fluorescence was monitored by an Olympus IX71 microscope (Olympus Co., Tokyo, Japan).