Cc1 buffer

Immunohistochemistry (IHC) reactions were performed on 4-μm paraffin sections with BenchMark XT device (Ventana/Roche). Primary antibodies were anti-CD3 (ready-to-use rabbit monoclonal antibody clone 2GV6, Ventana/Roche), anti-CD8 (rabbit monoclonal antibody clone SP57, Ventana/Roche), and anti-FOXP3 (mouse monoclonal antibody, clone 236A/E7; Abcam, Cambridge, UK at 1:200 dilution). For CD3 staining, the epitope retrieval was performed with CC1 buffer (Ventana/Roche) and the protocol used was the mild time (30 min) protocol; the antibody incubation time was 28 min at 37 °C. For CD8 staining, the epitope retrieval was performed with CC1 buffer mild time (30 min) protocol, the antibody incubation time was 32 min at 37 °C, and the ultraView amplification kit (Ventana/Roche) was used with 4-min incubation. For FOXP3, staining was performed according to the manufacturer’s instructions. Signal detection was performed with the ultraView universal DAB Detection Kit (Ventana/Roche). CD8 and CD3 membrane staining was considered positive, while identification of FOXP3-positive T lymphocytes was based on distinct nuclear expression.

For xenograft samples and pancreatic tissue from KPC mice, dissected tissues were fixed immediately after removal in 10% buffered formalin solution for a maximum of 24 h at room temperature before being dehydrated and paraffin-embedded under a vacuum. The tissue sections were deparaffinized with EZ Prep buffer (Ventana Medical Systems, Santa Clara, USA). Antigen retrieval was performed with CC1 buffer (Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Lincoln, RI, USA). IHC detection was performed using the Discovery XT processor (Ventana Medical Systems). All tumor tissues were harvested from mice and fixed in 4% PFA overnight. Fixed tissues were dehydrated, embedded in paraffin, and sliced into 3-µm sections. The tissue sections were deparaffinized with EZ Prep buffer, and antigen retrieval was performed with CC1 buffer and heat treatment in citrate buffer at pH 6.0 (Ribo CC, Ventana Medical Systems). Tumor sections were incubated with the indicated primary antibodies and detection was performed with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions, followed by counterstaining with hematoxylin (Ventana Medical Systems). Images were obtained using Vectra Polaris (PerkinElmer).

Discovery-Ultra was used for IHC analysis of CTLA-4 expression. Mouse monoclonal CTLA-4 (CD-152) antibody, clone 14D3, (eBioscience, cat#14-1529-80), was used in this study. Antibody concentrations and unmasking pretreatments was tested using both TMA and whole tissue slides. Positive and negative tissue controls, and negative subclass isotype-matched control antibody (Biolegend, cat#400203) was included.
Deparaffinization was performed in EZ Prep buffer (Roche, 5279755001) at 68 °C (3 × 12 min). Target unmasking to disengage cross linking effect of formalin fixation was done at 95 °C with CC1 buffer (Roche, 6414575001) for 24 minutes. Endogenous peroxidase was blocked for 8 minutes by Discovery inhibitor CM (Roche, 05266645001). Mouse monoclonal CTLA-4 antibody (1:100 dilution) was incubated for 32 minutes at 36 °C. Secondary multimer antibody OmniMap anti-Ms HRP (Roche, 5269652001) followed as immunologic detection for 16 minutes and substrate reaction was done with Discovery ChromoMap DAB detection kit (Roche, 526645001). All sections were counterstained for 16 minutes with hematoxylin (Roche, 5266726001) and post counterstained for 4 minutes with bluing Reagent, (Roche, 5266769001).