Dab substrate kit

Tumor tissues underwent paraffin embedding, prior to sectioning at 4 μm, followed by treatment with antibodies (Supplemental Table 4) at 1:200–300 dilution, and immunostaining, as instructed in a standard protocol, using the DAB Substrate Kit (ZSGB-BIO). Cell proliferation was determined by quantifying Ki-67 staining (percentage of positive cells), in the presence of an internal or intra-assay control. The Ki-67 protein levels received scores of 0, 1, 2, 3, which represented negative, weak positive, positive, and strong positive, respectively. Scores of 1, 2, 3 were considered positive, and the percentage of positive staining cells were determined. The protein levels were determined via integral optical density (IOD) using Image-pro plus 6.0 (Media Cybernetics, USA). We also stained xenograft tumors with hematoxylin and eosin (H&E), and assessed under the same magnification, light brightness, and exposure intensity.

With approval from the Ethics Committee, PDAC samples were obtained from 85 patients (aged 31 years to 73 years) undergoing surgical resection with histologic diagnosis of PDAC at the Tianjin Medical University Cancer Institute and Hospital. Another cohort was 76 PDAC patients undergoing surgical resection and Gem treatment at the Tianjin Medical University Cancer Institute and Hospital. Immunohistochemistry for IL-37, HIF-1α and p-STAT3 (705) of PDAC patient tissues was performed using a DAB substrate kit (ZSGB-BIO, Beijing, China). Immunoreactivity was semi-quantitatively scored according to the estimated percentage of positive tumor cells as previously described 21 (link). Staining intensity was scored 0 (negative), 1 (low), 2 (medium), and 3 (high). Staining extent was scored 0 (0% stained), 1 (1%-25% stained), 2 (26%-50% stained), and 3 (51%-100% stained). The final score was determined by multiplying the intensity scores with staining extent and ranged from 0 to 9. Final scores (intensity score × percentage score) less than 2 were considered as negative staining (-), 2-3 were low staining (+), 4-6 were medium staining (++) and > 6 were high staining (+++).

IHC was carried out on 5 μm sections of paraffin-embedded tissue. After slides baking, dewaxing, rehydration, the antigen retrieval was performed. The primary antibodies used for IHC were anti-cleaved caspase 3 (#9664, Cell Signaling Technology, Massachusetts, USA) and anti-Ksuc (PTM401, PTMBio, Hangzhou, China) diluted 1:100 in 5% bovine serum albumin (BSA) (SW3015, Solarbio). The secondary antibody and visualized stain for peroxidase was performed using the DAB Substrate Kit (PV-6000D, ZSGBbio, Beijing, China), and slides were counterstained with hematoxylin (AR1180-100, Boster). To confirm the validity and reliability of reagents used in IHC staining especially the specific antibody, the negative control groups were set (Fig. S2A).