Formalin-fixed, paraffin embedded (FFPE) tumor tissues were obtained from 4 study dogs (all study dogs that had tissue samples available) and from a reference group of 14 dogs with untreated gliomas. Immunohistochemistry was performed on FFPE tissues using a Leica BOND-MAX Fully Automated IHC Staining System, with canine cross-reactive antibodies: mouse monoclonal anti-human CD3 (Leica Biosystems, Lincolnshire, IL, Cat# PA0554, RRID:AB_10554454, clone LN10) and mouse monoclonal anti-human Myeloid/Histiocyte antigen (Bio-Rad Cat# MCA874GA, RRID:AB_324314, clone MAC387). Antigen retrieval was performed using Leica Epitope Retrieval Solution 2 (Tris-EDTA buffer, pH 9) for 20 minutes at 95°C. Detection was performed with PowerVision IHC Detection Systems (Leica Biosystems, Inc.), using a polymeric horseradish peroxidase anti-mouse IgG and DAB chromogen (CD3) or a polymeric alkaline phosphatase anti-mouse IgG and Fast Red chromogen (MAC387).
Whole slide brightfield images of IHC stained slides were digitally captured using an Olympus VS120 slide scanner at 20x magnification and fixed exposure times for all samples. Quantitative image analysis was performed using Visiopharm software (Hesholm, Denmark) and visually confirmed by a veterinary pathologist (DPR). The density of tumor infiltrating immune cells was calculated as number of immune cells per mm2 of viable tumor tissue.
Whole slide brightfield images of IHC stained slides were digitally captured using an Olympus VS120 slide scanner at 20x magnification and fixed exposure times for all samples. Quantitative image analysis was performed using Visiopharm software (Hesholm, Denmark) and visually confirmed by a veterinary pathologist (DPR). The density of tumor infiltrating immune cells was calculated as number of immune cells per mm2 of viable tumor tissue.