Nbp1 87402

Immunohistochemical detection of epithelial tight junction (TJ) proteins: ZO-1, occludin and claudin-1 was performed using a Rabbit specific HRP/DAB (ABC) Detection IHC kit (ab64261, Abcam, Australia) following the manufacturer’s instructions and as previously described [19 (link)]. Antibodies anti-ZO-1 (NBP1-85046, Novus Biologicals, Australia, 1:400); anti-occludin (NBP1-87402, Novus, 1:600) and anti-claudin-1 (NBP1-77036, Novus, 1 μg/mL) were used for incubating the colonic sections overnight at 4 °C. Computer-assisted image analysis was performed with a Leica DM500 microscope (Leica Microsystems, Wetzlar, Germany), Leica ICC50 W camera (Leica Microsystems, Wetzlar, Germany), and Image Pro Plus 7.0 (Media Cybernetics, Inc., Rockville, MD, USA) software. The expression of tight junction (TJ) proteins: ZO-1, occludin and claudin-1 was blindly assessed by choosing random five fields on each slide (n = 4/group). Barrier TJ protein expressions and staining intensity in colonic epithelium were expressed as the percentage expression of a respective TJ protein.

Total proteins were extracted from the abdominal aorta of mice or from MVECs using RIPA lysis buffer containing protease and phosphatase inhibitors. The abdominal aorta was crushed using an ultrasonic cell pulverizer (scientz‐IID, scientz) to obtain the supernatant (proteins). Next, the protein concentration was quantified using a BCA assay kit. A 10% SDS‐PAGE gel was used to separate the protein. Then, the protein was transferred to polyvinylidene fluoride membranes. After being blocked with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The membrane was then incubated with secondary HRP‐conjugated antibody for 1 h after being washed 10 min/each for three times with TBST buffer. Blots were then developed using a chemiluminescent kit according to the manufacturer's instructions. Antibodies against the following proteins were employed: VEGF (1:1000, #ab32152, Abcam), Angiopoietin 1 (Ang‐1) (1:1000, #ab 183701, Abcam), Occludin (1:500, #NBP1‐87402, Novus), VE‐cadherin (1:1000, # sc‐9989, Santa Cruz), PES1 (1:500, #NBP2‐55211, Novus) and β‐actin (1:5000, # A5441, Sigma), respectively.