Dab substrate

Paraffin blocks of the left lobe of the lung were sectioned, and hydrogen peroxide was used to quench endogenous peroxidase activity. Sections were immunostained with antibodies against CD206 (1:200, Abcam), CD8 (1:40, 53–6.7, Biolegend), cleaved caspase 3 (1:100, 5A1E, Cell Signaling), PCNA (1:200, sc-56, Santa Cruz Biothechonlogies), F4/80 (1:50, BM8, Invitrogen), CD107a (1:50, Biolegend), p-ERK (1:50, Cell Signaling) and visualized with biotinylated anti-rabbit or anti-rat secondary antibodies (Cell Signaling or Vector Labs), as previously described^{53 (link)}. 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 mice was fixed in 10% phosphate-buffered formalin for 48 hours, 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 α-SMA (1:50, 1A4, Abcam), F4/80 (1:40, BM8, EBioscience) or CD4 (1:40, Gk1.5, Biolegend), 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). Human microarrays were acquired from US Biomax, Inc. (PA485 and T141a) and stained for α-SMA was described above.

One-third of the tumor, spleen, and one mammary were removed from female MMTV-Neu and PyMT mice and fixed in 10% phosphate-buffered formalin for 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 CD206 (1:200, Abcam), Gr1 (1:40, BM8, R&D), CD8 (1:40, 53–6.7, Biolegend), FOXP3 (1:50, FJK-16s, EBiosciences), PD-1 (1:100, EPR20665, Abcam), PD-L1 (1:50, MIH6, Abcam; R&D 1:50 for dual staining), cleaved caspase 3 (1:100, 5A1E, Cell Signaling), PCNA (1:200, sc-56, Santa Cruz Biothechonlogies), p-STAT1 (1:50, Cell Signaling), F4/80 (1:50, Abcam) 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). Dual staining was performed with a Double Stain IHC kit using AP-rabbit and HRP-rat antibodies (Abcam, ab183285).

Fresh BAT was fixed in formalin then paraffin-embedded for sectioning and staining with hematoxylin and eosin. For F4/80 staining, BAT was frozen in OCT prior to sectioning and F4/80 staining. Reagents include: F4/80 primary antibody (1:400 AbdSerotec, MCA497G), anti-rat IgG (Vector, MP-7444), DAB substrate (Cell Signaling Technology), Mayer’s hematoxylin (Sigma). Images were acquired using a Nikon Eclipse Ti Intensilight microscope at 40x magnification.

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).

Formalin-fixed tissues were embedded in paraffin and sectioned by the Histology Core. Boiling citrate buffer was used for antigen retrieval, and endogenous peroxidase activity was quenched using hydrogen peroxide. Tissue sections were stained with antibodies against IL-18 (1 μg/mL, PA5-79481, Thermo Fisher Scientific), and Col6a3 (20 µg/mL, PA5-49914, Thermo Fisher Scientific), as described [34 (link)]. Sections were then labeled with biotinylated secondary antibodies (anti-rabbit, Cell Signaling Technology, Danvers, MA, USA; anti-rat, Vector Labs, Burlingame, CA, USA), as previously described. [34 (link)] A DAB substrate (Cell Signaling) was used for signal detection, as per manufacturer-provided protocols, and sections were counterstained with hematoxylin (Vector Labs). The Fiji ImageJ image processing package (version ImageJ2) was used for quantification of intensity of DAB staining by the color deconvolution method [53 (link)] and mean gray value was used to calculate optical density by the formula OD = log (max intensity/mean intensity, with a maximum intensity of 255 for 8–bit images [54 (link)].

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′).