Ab239074

The tissue microarray (TMA) incorporated tissue sections that were preserved through formalin fixation and paraffin embedding. Haematoxylin and eosin staining was then performed to analyse the histological attributes of the samples. Each sample, measuring 1.5 mm in diameter, was orderly arranged onto slides.
For immunohistochemistry (IHC), slides were deparaffinized and pre‐treated in EDTA buffer (pH 9.0). Primary anti‐STING1 antibody (Abcam, ab239074, 1:1000) or anti‐SMAD4 antibody (Abcam, ab40759, 1:100) was applied and incubated at 4°C overnight. Goat anti‐rabbit antibody (Zsbio) was applied at room temperature for 30 min. Then slides were incubated with 3,3‐diaminobenzidine (DAB) solution (Zsbio).
The IHC score of tumour areas was quantified by QuantCenter software.²⁹ H score method was performed to analyse the expression of STING1 and SMAD4. The cut‐off point was determined by the receiver operating characteristic (ROC) curve according to a previous study.³⁰

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.