Biotinylated anti rabbit or anti rat secondary antibodies

A third of the tumor from MMTV-neu or the left lung from A/J mice were fixed in NBF for 48 h, embedded in paraffin, and sectioned. Endogenous peroxidase was quenched by hydrogen peroxide. Sections were immunostained with the following antibodies: CD206 (1:200, Abcam, Cambridge, United Kingdom), CD4 (1:40, GK1.5, Biolegend, San Diego, CA, USA), CD8 (1:40, 53–6.7, Biolegend, San Diego, CA, USA), FOXP3 (1:50, FJK-16s, EBiosciences, San Diego, CA, USA), PD-1 (1:100, EPR20665, Abcam, San Diego, CA, USA), p-ERK (1:100, 5A1E, Cell Signaling, Danvers, MA, USA), PCNA (1:200, sc-56, Santa Cruz Biotechnologies, Dallas, TX, USA), and F4/80 (1:50, Abcam, Cambridge, United Kingdom) and visualized with biotinylated anti-rabbit or anti-rat secondary antibodies (Cell Signaling or Vector Labs). Signal was detected using a DAB substrate (Cell Signaling) following the manufacturer’s recommendations. Sections were counterstained with hematoxylin (Vector Labs).

One third of the pancreas and spleen removed from KC or KPC mice was fixed in 10% phosphate-buffered formalin for at least 48 h, embedded in paraffin blocks and sectioned (5–6 µm). Hydrogen peroxide was used to quench endogenous peroxidase activity. Sections were immunostained with antibodies raised against Cytokeratin 19 (1:400, Abcam), FOXP3 (1:50, FJK-16s, EBioscience), PD-L1 (1:50, MIH6, Abcam), CD4 (1:40, Gk1.5, Biolegend), or CD45 (1:100, 30-F11, BD Pharmingen, San Diego, CA, USA) and visualized with biotinylated anti-rabbit or anti-rat secondary antibodies (Cell Signaling or Vector Labs, Burlingame, CA, USA). Signal was detected using a DAB substrate (Cell Signaling) following the manufacturer’s recommendations. Sections were counterstained with hematoxylin (Vector Labs). For RT-PCR cells were collected and RNA was extracted using TRIzol (Thermo Fisher, Grand Island, NY, USA) following the manufacturer’s instructions. Two micrograms of RNA were reverse transcribed, and 1 μL of complementary DNA from this reaction was added to 12.5 μL of iQ SYBRGREEN Supermix (Bio-Rad, Hercules, CA, USA), 1 μL of validated RT2 quantitative PCR (qPCR) PD-L1 (F: 5′-TGC GGA CTA CAA GCG AAT CAC G-3′; R: 5′-CTC AGC TTC TGG ATA ACC CTC G-3′), GAPDH (F: 5′-GGAGCGAGATCCCTCCAAAAT-3′; R: 5′-GGCTGTTGTCATACTTCTCATGG-3′).