Bs 3589r

The thrombus-containing IVC, fixed with 4% paraformaldehyde for 24 h, were embedded in paraffin and sectioned into slices (5 μm). After dewaxing with xylene, the slices were dehydrated using ethanol. The sections were then treated with citrate buffer (pH 6.0, m053201, mreda, Beijing, China) at 95°C for 10 min and sealed with 5% bovine serum albumin (BSA) (A8010, Solarbio, Beijing, China) for 60 min at 25°C. Thereafter, sections were incubated with rabbit anti-phospho-eNOS (Thr113) (1:200, bs-3589R, Bioss, Beijing, China) at 4°C overnight. After washing with PBS, slices were incubated with a second antibody, lgG, labeled with Cy3 (rabbit anti-rat lgG/Cy3, 1:1000, bs-0293R, Bioss, Beijing, China) for 60 min at room temperature (25°C). After washing with PBS, sections were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. The fluorescent images were obtained using a laser confocal microscope (LSM800, Zeiss, Germany) and analyzed using Image J software (version 6; National Institutes of Health).

Cells were lysed on ice with RIPA buffer (P0013B, Beyotime, Shanghai, China), and the supernatant was collected after centrifugation. Then, 10% SDS-PAGE gel electrophoresis was carried out, followed by being transferred to a PVDF membrane. After a 2-h block in a western blotting buffer (5% non-fat milk), the membrane was incubated overnight in a shaker with primary antibody dilution. The primary antibodies used specifically were OPN (bs-23258R, rabbit, 1:1000, Bioss, Beijing, China; ab63856, rabbit, 1:1000, Abcam, Cambridge, UK), Orai1 (ab111960, rabbit, 1:1000, Abcam, Cambridge, UK; 28411-1-AP, rabbit, 1:500, Proteintech, Wuhan, China), STIM1 (11565-1-AP, rabbit, 1:1000, Proteintech, Wuhan, China), eNOS (bs-20608R, rabbit, 1:1000, Bioss, Beijing, China), p-eNOS (bs-3589R, rabbit, 1:1000, Bioss, Beijing, China), and GAPDH (TA-08, mouse, 1:2000, Zsbio, Beijing, China). Afterward, HRP-conjugated secondary antibodies were diluted in a secondary antibody dilution solution, specifically goat anti-rabbit (ZB-2301, Zsbio, Beijing, China) and goat anti-mouse (ZB-2305, Zsbio, Beijing, China) at a ratio of 1:20000. After 2 h of incubation at room temperature, an ECL detection kit (340958, Thermo, Massachusetts, USA) was used to detect the proteins. The density quantification of the immunoblot was carried out with a chemiluminescent gel imaging analysis system (Bio-Rad, California, USA).