3 3 diaminobenzidine dab solution

To stain fibroblast-related proteins, the cells were permeabilized with 0.1% Triton X-100 and then incubation with the following primary antibodies overnight at 4 °C: anti-vimentin (1:200, ZSGB-Bio, Beijing, China), anti-fibroblast specific protein 1 (FSP-1) (1:200, ZSGB-Bio), anti-α-smooth muscle actin (α-SMA) (1:200, ZSGB-Bio) and anti-cell keratin 5/6 (CK5/6) (1:200, ZSGB-Bio). The cells were incubated with biotinylated secondary antibodies (1:4000, ZSGB-Bio) for 2 h at room temperature. 3,3-Diaminobenzidine (DAB) solution (ZSGB-Bio) was then used to visualize the localization of the target proteins. Finally, the cells were counterstained with haematoxylin (ZSGB-Bio) for nuclear staining. PBS was used as the negative control. The slides were observed with an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

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