Ab190085

The liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned into 4 μM thick slices. Hematoxylin and eosin (H&E) staining, reticular fiber staining, and Masson staining were performed according to the standard dyeing scheme. Immunohistochemistry was used to measure the levels of hepatic CK18 (Servicebio, GB11232), colorectal Zonula occludens protein 1 (ZO-1) (Abcam, ab190085), and occludin (Abcam, ab216327).

The western blotting assay was performed as described earlier (Xu et al., 2017 (link)). Rats were euthanized 24 h after SAH, and the brains were obtained by decapitation after perfusion. A small amount of tissue was obtained from the ipsilateral basal cortex, weighed, lysed, homogenized, and centrifuged. The bicinchoninic acid method was used for protein quantification. An equal amount of protein samples (60 μg) was added to sodium dodecyl sulfate-polyacrylamide gels for vertical electrophoresis, and the samples were electro-transferred to polyvinylidene difluoride membranes. The membranes were blocked using skim milk for 30 min, and the following primary antibodies against ZO-1 (1:1,000, ab190085, Abcam, United States), occludin (1:1,000, ab216327, Abcam, United States), HIF-1α (1:2,000, ab179483, Abcam, United States), VEGF (1:1,000, ab214424, Abcam, United States), MMP-9 (1:5,000, ab76003, Abcam, United States), and GAPDH (1:5,000, ab8245, Abcam, United States) were added. The membranes were incubated at 4°C overnight, washed with TBST, and secondary antibodies were added dropwise before incubating for 60 min at room temperature. The membranes were washed, exposed, developed, and imaged utilizing a gel imaging system, and the ImageJ software (NIH) was used for quantitative analysis.

The total protein in the lung tissue was extracted with cell lysis buffer and measured using a BCA protein assay kit, and the proteins were resolved using SDS–polyacrylamide gel electrophoresis. Electrophoresis of proteins was followed by transfer to PVDF membrane. The membrane was incubated with primary antibodies against Bax (ab32503, Abcam, China; 1 : 1000), Bcl-2 (ab32124, Abcam, China; 1 : 1000), cleaved caspase-3 (ab32042, Abcam, China; 1 : 1000), SDC-1 (ab128936, Abcam, China; 1 : 1000), claudin-5 (ab131259, Abcam, China; 1 : 1000), ZO-1 (ab190085, Abcam, China; 1 : 1000), and VE-cadherin (ab231227, Abcam, China; 1 : 1000) at 4°C overnight, followed by 1 hour at room temperature with the secondary antibody. The protein bands were identified using an enhanced detection chemiluminescence (ECL) technique.

Segments of distal ileum were fixed with formalin, embedded in paraffin, sectioned into 4-μm-thick slices, and subjected to immunohistochemistry (IHC) to detect ZO-1 and occludin. The sections were deparaffinized, rehydrated, and incubated with hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. After incubation for 20 min in normal goat serum, sections were immunostained using goat anti-ZO-1 polyclonal primary antibody (ab190085; Abcam, Cambridge, UK) or rabbit anti-occludin polyclonal primary antibody (ab31721; Abcam, Cambridge, UK) together with a streptavidin-biotin-peroxidase detection system and a DAB reagent kit (Zhongshan Jinqiao Biotechnology Company, Beijing, China). The sections were then stained by hematoxylin, thoroughly washed, dehydrated, and mounted using coverslips and DPX mounting medium. The slides were observed under a microscope (#BX51T-PHD-J11, Olympus Corporation, Tokyo, Japan). The number of immunopositive cells in 5 fields of view was counted independently by two researchers who were blinded to the grouping. The number of positive cells (value A) was graded as 0 (0–1%), 1 (1–10%), 2 (10–50%), 3 (50–80%), or 4 (80–100%). The staining intensity (value B) was graded as 0 (negative), 1 (weakly positive), 2 (positive), or 3 (strongly positive). The IHC score was then calculated as IHC = A × B (34 (link)).

Aortic wall samples from the patients and mouse aortic wall tissues were fixed with 4% paraformaldehyde for 24 hours and then embedded in paraffin and sliced into 4 μm sections. The morphology and fibrosis of the aortic wall were determined using H&E staining and picrosirius red staining (PSR) according to the manufacturer's instructions. Immunohistochemical staining was performed with antibodies against NAMPT (Abcam, ab236874) and SIRT1 (Abcam, ab110304). Immunofluorescence staining was performed using antibodies against CD31 (Abcam, ab28364), NAMPT (Abcam, ab236874), and ZO-1 (Abcam, ab190085) using previously described methods. Images were acquired and analyzed using Image-Pro Plus 6.0 software as previously described [25 (link)].

HUVECs were stimulated with 0.4 μg/mL NETs for 24 h. The sample was removed from the 24-well plate, fixed with 1% paraformaldehyde for 15 min, washed twice with PBS, incubated with 3% bovine serum albumin for 30 min, and then washed twice with PBS. The primary antibodies, 1:1000 dilutions of anti-ZO-1 (ab190085; Abcam), anti-VE (ab33168; Abcam), anti-CD31 (ab228968; Abcam), anti-ANGPT2 (Df6137; Affinity, China), anti-von Willebrand factor (vWF) (ab154193; Abcam), or anti-CCDC25 (21,209–1-AP; Proteintech), were incubated overnight at 4 °C. The secondary antibodies, labeled with Alexa Fluor 488 and 594 (1:200; Abcam, USA), were incubated for 30 min and washed twice with PBS. Cytoskeletal staining was conducted as follows: the ghost pen cyclopeptide was incubated for 10 min and washed twice with PBS. Nuclear staining was performed via incubation with DAPI (Solarbio Life Science) for 10 min, followed by washing twice with PBS. Sharp tweezers were used to remove the slide from a 24-well plate and affix the cellular side upside down onto the glass slide. Glycerol was added for anti-quenching, and the slides were observed and photographed using a confocal microscope.