Bs 10412r

For the immunofluorescence, eye sections were deparaffinized and rehydrated before being incubated overnight in Epitope Retrieval Solution (Leica Biosystems Inc., Buffalo Grove, IL, USA) at 60 °C. Then, the sections were blocked at room temperature for 40 min with a solution containing PBS, 2% bovine serum albumin (BSA) (ABIN934476, Antibodies-online), and 0.1% Triton-X100 (X100, Sigma-Aldrich, St. Louis, MO, USA). Primary rabbit anti-VEGFR2 (bs-10412R, 1:100 dilution; Bioss, Woburn, MA, USA) and rabbit anti-TACE (bs-4236R; 1:100 dilution; Bioss, USA) antibodies were diluted in the primary antibody diluting buffer (Bio-Optica, Milano, Italy) and incubated for 2 h at room temperature. Slides were incubated for 30 min at room temperature in the dark with Cy5-labeled goat anti-rabbit IgG secondary antibody (A10523; Thermo Fisher Scientific Inc., Rockford, IL, USA) and diluted 1:500 in PBS before being counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. CC/Mount aqueous mounting medium (Sigma-Aldrich, St. Louis, MO, USA) was used to mount the slides. Images were acquired using a Leica TCS SP8 laser scanning confocal microscope. The percentage of positive-stained area/total area was quantified using ImageJ software.

Rat lung ECs were isolated from lung single cell suspensions of naive male Sprague Dawley rats (Envigo). Rat lungs were removed, and a single cell suspension was prepared from the peripheral lung tissue using a modification of the protocol by van Beijnum et al. [30 (link)]. In brief, tissue was minced into <1mm³ pieces, and digested in a solution of 0.1% collagenase II and 2.5 U/ml dispase solution (both from Thermo Fisher Scientific, Waltham, MA) for 30 min at 37˚C. Then, tissue pieces were incubated with 0.1% DNase (Sigma-Aldrich, St. Louis, MO) for 30 min at 37˚C. CD31⁺ cells were obtained by immunomagnetic sorting using magnets and the “Any Species positive selection” kit from Stem Cell Technologies (Vancouver, BC) and CD31 antibody from R&D Systems (FAB3628). ECs were cultured on type I collagen coated dishes with EGM-2MV medium (Lonza, Walkersville, MD) and characterized by flow cytometry to identify EC markers CD144 (VE-cadherin, bs-0878R, Bioss antibodies, Woburn, MA) and vascular endothelial growth factor receptor 2 (VEGFR2, bs-10412R, Bioss). Rat lung ECs were used in passages 2–4. Expression of myeloid/hematopoietic markers CD133 (bs-0209R, Bioss) and CD11b/c (554862, BD Biosciences) was excluded by flow cytometry.

Cellular proteins were extracted with RIPA lysis buffer (Sigma Aldrich), separated using SDS-PAGE (Beyotime), and transferred to polyvinylidene fluoride membranes (Beyotime). Membranes were then blocked in 5% skim milk and treated with anti-E2F7 (1 : 1000, ab56022; Abcam, UK), VEGFR2 (1 : 300, bs-10412R; Bioss, China), p-VEGFR2 (1 : 1000, ab5473; Abcam, UK), GAPDH (1 : 3000, bs-2188R; Bioss, China), and mouse anti-rabbit secondary antibody (1 : 5000, bs-0295M-HRP; Bioss, China). Western blots were visualized using the ECL system (Beyotime).