Vs120 scanner

Picosirius red (PSR) staining was used for analysis of dermal collagen bundle alignment, according to the schematic shown in (Figure A inS1 File). Whole slide imaging was performed using both brightfield and polarized light microscopy at 40X magnification, using an Olympus VS120 scanner (Olympus Corp, Tokyo, Japan). Slides were digitized and individual collagen bundles identified using VisioPharm morphometry software (Ver 5.0.2, Copenhagen, Denmark). Analysis of dermis thickness (defined as area divided by contour length), total dermal collagen (measured as fraction of dermis with PSR staining), and bundle alignment was performed by scripting in Python and R.

To evaluate CD31⁺ vessels in tumours, 1.4 × 10⁴ B16 mouse melanoma cells were injected into the right flanks of age-matched, syngeneic (C57Bl/6 N) Dsg2^+/+ (WT) and Dsg2^lo/lo mice and resultant tumours harvested at endpoint. Sections were cut from FFPE-stored tumour tissues and labelled with anti-CD31 antibody (BD Biosciences). Secondary detection was performed using biotinylated anti-rat-Ig, streptavidin-conjugated horse-radish peroxidase and 3-amino-9-ethylcarbazole chromogen. Stained sections were scanned using an Olympus VS120 scanner and CD31⁺ blood vessels were counted in each field to obtain an average number of CD31 + blood vessels per field.

FFPE OP tissues from three cases testing positive for FcaGHV1 DNA on OP swabs, and three testing negative, were processed for ISH, as described previously (Aghazadeh et al. 2018) [21 (link)]. Briefly, colorimetric ISH was performed manually on 5 µm sections of FFPE tissue on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA) using the RNAscope 2.5 Red assay kit (322360, Advanced Cell Diagnostics, Inc., Hayward, CA, USA) and the ISH probe V-FcaGHV1-ORF50 (510481, ACD). Each section was pretreated with heat and protease prior to probe hybridization for 2 h at 40 °C. Negative controls used for validation of signal included a bacterial gene DapB probe and an uninfected animal. Slides were counterstained with hematoxylin and mounted with EcoMount (Biocare Medical, Concord, CA, USA). Slides were digitized using an Olympus VS120 scanner and a 40× objective with bright field illumination.

The day after the last administration of MRTX1257 (= D4 after RT), animals were sacrificed by cervical dislocation and subcutaneous tumors were harvested and then fixated using paraformaldehyde (PFA) 4%.
For detection of cleaved CD8, 4 µm sections were processed for heat-induced antigen retrieval (ER2 corresponding EDTA buffer pH9) for 20 min at 100 °C. Slides were incubated with the antibody (clone D4W2Z #98,941, 1: 400, Cell Signaling) for 1 h at room temperature. Then, the slides were incubated with the Rabbit HRP PowerVision Kit (Leica Biosystems, #PV6119). The signal was revealed with diaminobenzidine (DAB).
For Ki67, slides were incubated with a monoclonal rabbit anti-Ki67 antibody (Cell Signaling, 1: 500). The signal was revealed with the Rabbit PowerVision Kit (UltraVision Technologies).
All slides were scanned at 20X using VS120 scanner (Olympus, Japan). Images were processed and staining quantified using QuPath v0.4.0 [27 (link)]. Regions of interest were manually delineated. Inside these regions, tissue was automatically detected using a trained classifier. Color deconvolution (Hematoxylin, DAB and residual) was applied to the images. Cells were detected based on their optical density and positivity (CD8⁺ or Ki67⁺) was assessed based on the amount of DAB signal in each cell. Results were expressed as a number of stained cells by square millimeter of analyzed tissue.

Paraffin sections of 34 WHO grade IV glioma samples, provided by the Australian Genomics and Clinical Outcomes of Glioma (AGOG) tissue bank, were used for this study (University of Sydney Human Ethics Committee Project number 2016/027). Paraffin sections were stained with H&E and scanned at 40X magnification using an Olympus VS-120 scanner. Adjacent sections were processed for CD276 immunohistochemistry at St. Vincent’s Hospital, Sydney, and at the Department of Neuropathology of Royal Prince Alfred Hospital, respectively.

In situ hybridization (ISH) on 4 μm-thick mouse or human kidney sections was performed using the RNAscope^® 2.5 HD Duplex Assay from Advanced Cell Diagnostics (California, USA) and the DDR1 probes according to the manufacturer’s instructions: mouse DDR1 (RNAscope^® Probe-Mm-Ddr1-C2) and human DDR1 (RNAscope^® Probe- Hs-DDR1-C2, target region 285—2016, #593591-C2). Double ISH was performed on mouse kidney sections using mouse DDR1 probe and mouse Acta2 (RNAscope^® Probe- Mm-Acta2-C2) or mouse Emr1 (RNAscope^® Probe- Mm-Emr1). Images were acquired with an Olympus VS120 scanner (Olympus AG, Switzerland) equipped with a VC50 camera and 20× objective.

We designed an antisense probe, V-FeHepadnavirus, (Advanced Cell Diagnostics, Inc., Hayward, CA, USA) targeting region 604–1477 of DCH, Genbank accession number MH3079301 (Figure 1) [4 (link)]. Cases that tested positive for DCH by cPCR, and negative control tissues, were progressed to ISH. Colorimetric ISH was performed manually on 5 μm sections of FFPE tissue on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA) using the RNAscope 2.5 Red assay kit (Advanced Cell Diagnostics, Inc.). Each section was pretreated with heat and protease prior to probe hybridization for 2 h at 40 °C.
For signal validation, negative control probes were used to probe serial sections, including a probe designed to detect dihydrodipicolinate reductase (DapB) of Escherichia coli (all cases). Uninfected, histologically normal feline liver tissue served as a negative tissue control (n = 3). Slides were counterstained with hematoxylin and mounted with EcoMount (Biocare Medical, Concord, CA). Slides were digitized using an Olympus VS120 scanner and a 40× objective with bright-field illumination.