Cc1 antigen retrieval buffer

Immunohistochemistry (IHC) was performed in cut sections of pancreatic tumors using anti-MUC1 monoclonal antibody (MAb) clone 014E (MAb MUC1/014E, the kind gift of Suguru Yonezawa) [38 (link)] and anti-MUC4 MAb clone 8G7 (MAb MUC4/8G7, the kind gift of Surinder K. Batra) [39 (link)] using the immunoperoxidase method. Antigen retrieval was performed using CC1 antigen retrieval buffer (pH 8.5, EDTA, 100°C, 30 minutes; Ventana Medical Systems, AZ, USA) for all sections. Following incubation with the primary antibodies (MAb MUC1/014E diluted 1:5, 37°C, 32 minutes; MAb MUC4/8G7 diluted 1:3000, 37°C, 32 minutes) in phosphate buffered saline, pH 7.4 (PBS) with 1% bovine serum albumin (BSA), sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (UltraView DAB, Ventana Medical Systems). The control staining (normal mouse serum or PBS-BSA instead of the primary antibodies) showed no reaction.

Immunohistochemistry (IHC) was performed in cut sections of lung tumors using anti-MUC4 MAb clone 8G7 (MAb MUC4/8G7, the kind gift of Surinder K. Batra) [9 (link)] using the immunoperoxidase method. Antigen retrieval was performed using CC1 antigen retrieval buffer (pH 8.5, EDTA, 100°C, 30 minutes; Ventana Medical Systems, AZ, USA) for all sections. Following incubation in phosphate buffered saline, pH 7.4 (PBS) with 1% bovine serum albumin (BSA), sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (UltraView DAB, Ventana Medical Systems). The control staining (normal mouse serum or PBS-BSA instead of the primary antibodies) showed no reaction.

We selected 25 cases each that were predicted to have high and low immune response by using the value of the radiomic feature in the prognostic modules M2, M9, and M12 that showed the highest absolute correlation to the mean expression of genes in the CTLA4 inhibitory pathway that is supported to be associated with immune activity (Postow et al., 2015 (link); Pardoll, 2012 (link); Wolchok and Saenger, 2008 (link)). In total, 22 cases were available with enough tumor tissue and sufficient staining quality. Tumor cross section slides were stained using a Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ) as per manufacturer's protocol with recommended reagents. Briefly, slides were deparaffinized with EZ Prep solution (Ventana) and a heat-induced antigen retrieval method was used under mild cell conditioning using CC1 antigen retrieval buffer (Ventana). A rabbit primary antibody for CD3, (790–4341, Ventana) was used at supplied concentration and incubated for 16 min. Next a Ventana OmniMap Anti-Rabbit Secondary Antibody was applied to the samples for 16 min and the Ventana ChromoMap kit was used as the detection system. Slides were then counterstained with Hematoxylin and dehydrated. Finally, the slides were cover slipped as per normal laboratory protocol.