Nanozoomer whole slide scanner

Formalin-fixed paraffin-embedded mouse tumors from in vivo mouse experiments were subject to immunohistochemistry (IHC) analysis as conducted by Emory’s Cancer Tissue and Pathology Shared Resource. Sections from mouse tumors that did not render large enough tissue size were excluded from further downstream analyses. 4–5 µm slices were individually stained with hematoxylin (Sigma #517-28-2) and picrosirius red (Abcam #ab150681), and primary antibodies (table 1) were developed using DAB (3,3′-diaminobenzidine) substrate (Vector Laboratories #SK-4100). Slides were scanned using an Olympus Nanozoomer whole slide scanner. Images were analyzed using Qupath software (qupath.github.io). The total number of cells was quantified within a specified tissue area, and threshold detection was used to count the number of cells positive for a given antibody signal.29 (link) For picrosirius red, threshold detection was used to quantify the positive area (µm³) of the total tumor tissue area. Multiplex immunofluorescence staining included 4′,6-diamidino-2-phenylindole (DAPI) (PerkinElmer #CS1-0127-2ML), CD4, and CD8 primary antibodies (table 1), followed by Opal 690 and Opal 520 conjugated secondaries (PerkinElmer), respectively. Images were acquired using a Roche BenchMark ULTRA IHC/ISH System autostainer.