A1r confocal fluorescent microscope

Tumor sections were cut into 10µm sections using a CryoStat (Leica CM1850). Sections were fixed in acetone at −20C for 10 minutes then rehydrated with Phosphate Buffered Saline (PBS) for 5 minutes at room temperature. Sections were blocked with blocking buffer (PBS, 10% heat inactivated fetal bovine serum (FBS), and 0.1% Tween 20)) for 30 minutes. All sections were stained with antibodies for CD3 (Clone 17A2, Biolegend #100203), CD8 (Clone 53–6.7, Biolegend #100707), PD-1 (Clone RMP1–30, Biolegend #109111) and with 4’,6-diamidino-2-phenylindole (DAPI) in blocking buffer for 2 hours at room temperature. Samples were imaged using a Nikon A1R fluorescent confocal microscope. Settings for each series of images was determined using single color splenic controls and maintained throughout imaging. Composites of confocal images (4–6 planes per section) were made using maximum intensity projection in ImageJ and PD-1⁺ CTLs were counted if they stained positive for CD3, CD8, and PD-1. Six images were taken per tumor from three tumors per treatment condition across two independent experiments. All images were taken at 20x magnification (635.4µm by 635.4µm).

Tumors were rapidly frozen in O.C.T. compound and cut tumors into 15-20 μM sections using a Leica CM3050 S cryostat. Sections were placed in cold acetone for 10 minutes, rehydrated with Tris-buffered saline (TBS) for 20 minutes, and blocked with blocking buffer (TBS ⁺ 3% BSA and 0.1% Tween-20) for 20 minutes. Sections were stained in blocking buffer for one hour with antibodies from BioLegend specific for F4/80 (clone BM8), CD11b (clone M1/70), CD8α (clone 53-6.7), and CD45.2 (clone 104) as well as DAPI as indicated in the figure legends. Samples were imaged using the Nikon A1R fluorescent confocal microscope. Macrophage number per mm² image was calculated from the count of F4/80⁺, CD11b⁺ macrophages per image from multiple images per tumor and 2-3 tumors per treatment, as indicated in the figure legend. CD8⁺ T cell number per mm² image was calculated from the count of CD8⁺, CD45.2⁺, CD11b⁻, or CD8⁺, CD11b⁻ cells per image in multiple images per tumor and multiple tumors per treatment as indicated in the figure legends. Images were analyzed with ImageJ (https://fiji.sc/)(53 (link)).

For FISH staining, tissues were immediately fixed in a methacarn solution at 4°C overnight. Tissue cassettes were washed with PBS and put in 70% ethanol prior to paraffin embedding and sectioning. Sections were deparaffinized in xylene prior to 2 successive ethanol washes (95% and 90%) and rehydration with ddH2O. 16S Eubacteria and Hhep specific probes were added at 100nM in hybridization buffer (0.9M NaCl, 20mM Tris-HCl pH 7.2, 0.1% SDS) and left in a humidified chamber within a 56°C incubator overnight. A separate slide was stained with a ‘scrambled’ probe for control. Slides were washed with prewarmed wash buffer (0.9M NaCl, 20mM Tris-HCl pH 7.2) prior to DAPI/Hoechst staining. Slides were sealed with Prolong Antifade Gold and imaged using a Nikon A1R Confocal fluorescent microscope in the University of Pittsburgh Center for Biological Imaging.
For H&E images, tissues were butterflied and cleaned prior to fixing in Formalin overnight at room temperature in cassettes. Tissues were washed three times with PBS 24–72 hours, washed 3x in PBS, and stored in 70% ethanol prior to paraffin embedding. Following paraffin embedding, slides were stained for hematoxylin and eosin stains for morphological analysis, and imaged at a 10X magnification.