G1212

For hematoxylin and eosin (H&E) staining (G1005, Servicebio), tissues were fixed by 10% neutral formalin for 1 day, dehydrated and embedded in paraffin, and then cut into 5-µm-thick sections. After being dewaxed and treated with gradient alcohol, the sections were stained with hematoxylin for 5 min at room temperature and were washed with running water. Subsequently, alcohol solution with 1% hydrochloric acid and ammonia was employed. Subsequently, 1% eosin was utilized for further staining for 3 min at room temperature, gradient ethanol for section dehydration, and neutral balsam for section mounting, followed by microscope visualization (Nikon Eclipse E100; Nikon Corporation).
For IHC, the specimens were cut into sections (5-µm-thick). All tissue samples were fixed with 10% neutral formalin and embedded in paraffin. Paraffin sections were dewaxed and the endogenous peroxides quenched with 0.3% hydrogen peroxide. They were then incubated with anti-ENC1 antibody (1:200, ab124902; Abcam) overnight (at 4°C) and secondary antibody (1:200, 7074S; Cell Signaling Technology, Inc.) for 30 min (at 37°C). The sections were stained with diaminobenzidine (G1212; Servicebio). Images were collected under a microscope (Nikon Eclipse E100; Nikon Corporation).

As described above, the FFPE sections were deparaffinized and hydrated, and antigens were retrieved with Tris-EDTA buffer. Endogenous peroxidase activity was quenched with 3% H₂O₂. After blocking with goat serum, the slides were incubated with primary antibodies [major histocompatibility complex (MHC) I, ab134189/Abcam, 1:2000 or MHC II, ab170867/Abcam, 1:300], followed by incubation with HRP-conjugated secondary antibodies (AntGene, ANT020). Immunoperoxidase staining was performed using the 3,3′-diaminobenzidine (DAB) system (Servicebio, G1212). Finally, the slides were counterstained with hematoxylin and coverslipped with a mounting solution. The results were recorded using a TEKSQRAY scanner (SQS-40R) and quantified by three pathologists. The staining score was calculated as the area score × intensity score. Area scores were graded as 0–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4); intensity scores were graded as negative (0), weakly positive (1), positive (2), and strongly positive (3).

The Elivision Super HRP IHC Kit (KIT99-22, MXB, China) was used for immunohistochemistry according to the manufacturer’s instructions. Pancreatic sections were deparaffinized and rehydrated, and citrate buffer (pH 6.0, G1202, Servicebio, China) was used for antigen retrieval. Peroxidase blocking was performed in PBS with 3% H₂O₂ for 10 min, and permeabilization was conducted using 0.2% Triton X-100 (93443, Sigma–Aldrich, U.S.A.) for 45 min at room temperature. The slides were incubated with primary antibodies against IL-1β (1:200, GB11113, Servicebio), nuclear factor κ-B p65 (NF-κB p65) (1:200, GB11042, Servicebio), and Rbpj (1:200, 5313T, Cell Signaling Technology) overnight at 4°C. The slides were incubated with a response enhancer at 37°C for 20 min HRP-conjugated secondary antibody at 37°C for 20 min. AEC (ZLI-0936, ZSGB-Bio, China) or DAB (G1212, Servicebio, China) substrates were used for color development. Five sections in each group and ten 400× images per section were evaluated by calculating the number of positive cells/total number of cells.