Ifn β

Total protein was extracted from pancreatic, jejunal, and colonic tissues using membrane and cytoplasmic or nuclear and cytoplasmic protein extraction kits (Beyotime, Jiangsu, China) following the manufacturer's instructions. Equal amounts of protein for each sample were subjected to 5%–10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 and incubated with primary antibodies against TLR4, MyD88, TNF receptor associated factor- (TRAF-) 6, IL-1β, TRIF, TRIF-related adaptor molecule (TRAM), IFN-β (Bioss, Beijing, China), nuclear factor- (NF-) κB p65 (Cell Signaling Technology), interferon regulatory factor- (IRF-) 3 (Santa, Dallas, TX), claudin-1, occludin, ZO-1 (Sangon, Shanghai, China), β-actin, histone H3 (Cell Signaling Technology), or Na/K ATPase (Proteintech, Rosemont, IL) at 4°C overnight. Subsequently, membranes were washed before incubation with the corresponding secondary antibodies (Zhongshan Goldenbridge, Beijing, China) for 2 h at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Beyotime). Na/K ATPase, β-actin, and histone H3 served as internal controls for membrane proteins, cytoplasmic proteins, and nucleoproteins, respectively.

Individual tumor specimens were fixed in 10% of formalin and paraffin‐embedded. The tumor tissue sections (4 μm) were dewaxed, rehydrated, and subjected to antigen retrieval. The sections were incubated with specific primary antibodies against CD8 (Protintech, 1:800), granzyme B (Bioss, 1:100), IFN‐β (Bioss, 1:100) and glypican‐3 (Abcam, 1:200) at 4°C overnight. After being washed, the sections were reacted with HRP‐conjugated second antibodies and visualized with DAB. IHC signals were photoimaged (200 × magnification) under a light microscope (BX43 type, OLYMPUS). In addition, the frequency of apoptotic cells in tumor sections was measured by TUNEL assay using the TUNEL Detection Kit (ROCHE), according to the manufacture instructions.

Pre- and post-treatment tissue specimens were stained for STING (Cell signaling technology, #13647S, 1:100), PD-L1 (Bioss, bs-1103R, 1:300), IFN-β (Bioss, bs-23731R, 1:300), CD3 (Proteintech, 17617-1-AP, 1:100), CD8 (Proteintech, 66868-1-Ig, 1:1000). 5-um-thick tissue sections were deparaffinized in xylene, passed through graded alcohols, and antigen repaired with citrate buffer (pH=7.6) in a steam pressure cooker. The sections were blocked with 5% BSA 1H and incubated with primary antibodies at 4°C overnight, and then washed in 50 mM Tris-HCl, pH 7.4 and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoperoxidase staining was performed with DAB system. Slides were counterstained with haematoxylin. Immunofluorescence-stained sections were added dropwise with DAPI, incubated for 10 minutes and then rinsed 3 times with PBS. Immunohistochemical sections were dehydrated in in graded alcohol and xylene, and sealed with neutral gum. Immunostaining sections were sealed with glycerin.