Multispectral Immunophenotyping of Mouse Tumors

For multispectral immunophenotyping in mouse tumors, the murine-specific NEL810001KT Opal kit (Akoya, Marlborough, MA, USA) was used following the manufacturer’s instructions and as previously described (16 (link)), with additional markers. This kit includes the Alexa Fluor tyramides Opals 520, 570 and 690 and spectral DAPI. Opals 650 (R55503) was not included in the kit, so was purchased separately from Akoya. The following primary antibodies were used: anti-CD3 (1:150, ab16669; Abcam, Cambridge, United Kingdom), anti-CD4 (1:200, #25229S; Cell Signaling, Danvers, MA, USA), anti-CD8 (1:400, #98941S; Cell Signaling, Danvers, MA, USA) and anti-Id1 (1:1000, BCH-1/37-2; Biocheck, San Francisco, CA, USA). Vectra Polaris Automated Quantitative Pathology Imaging System and the Phenochart and InForm 2.4 software (Akoya, Marloborough, MA, USA) were used for sample scanning, spectral unmixing, and quantification of signals. Data were given as number of cells with a specific immunophenotype/total number of cells.