Ab228968

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.

Cells were seeded in 24-well plates (5 × 10⁴ cells/well) with the corresponding treatment. After fixation with 4% paraformaldehyde, the cells were permeabilized with 0.1% Triton X-100 for 5 min and then blocked for 30 min at room temperature. The cells were incubated with antibodies against F-actin (1:100, C2201S, Beyotime), TF (1:100, ab228968, Abcam), STING (1:100, ab239074, Abcam), TLR2 (1:150, MA5-32787, Invitrogen) overnight at 4 °C, and washed before incubated with fluorescent Alexa Fluor® 488-conjugated goat anti-mouse IgG (1:200, ab150113, Abcam) or Alexa Fluor® 594-conjugated goat anti-rabbit IgG (1:200, ab150080, Abcam). 4,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. To visualize NETs in lung tissue samples of the CLP mouse model, paraffin-embedded tissue sections were deparaffinized, rehydrated, and processed with heat induction for antigen retrieval. They were incubated with primary antibodies against citH3 (1:100, ab5103, Abcam), and MPO (1:100, ab90810, Abcam) after blocking, and DAPI was used to stain the nuclei. Images were taken under an Olympus microscope (Tokyo, Japan). The relative fluorescence intensity of targeted proteins was quantified using Fiji/ ImageJ software.

Immunoblotting for TF determination was performed as previously described (Eriksson et al., 2016 (link)). Briefly, total protein was extracted from samples, and the protein concentration was measured using BCA protein assay reagent (Beyotime, Shanghai, China). Then, the extracted protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by immunoblotting with specific antibodies against tissue factor (Abcam, ab228968).