Nel810001kt opal kit

The NEL810001KT Opal kit (Akoya, Marloborough, MA, USA) was used for multispectral immunophenotyping and multiplexed automatic quantification of CD3, CD4, and CD8 in tumors. Unspecific binding was blocked in Dako Antibody diluent (Dako, Glostrup, Denmark) for 10 minutes at RT. After antigen retrieval using a citrate buffer (pH = 6), samples were incubated with anti-CD8 (1:400, Cell Signaling) and then with secondary antibody and Opal-690 (1:100, Akoya) fluorochrome. Then, antigen retrieval was performed using EDTA (pH = 9) followed by anti-CD4 (1:1000, Abcam), secondary antibody, and Opal-570 (1:100, Akoya). Finally, antigen retrieval with citrate buffer (pH = 6) was applied before adding the anti-CD3 (1:75, Abcam), secondary antibody, and Opal-520 (1:100, Akoya). Ready-to-use secondary antibodies and Opals are provided by the kit. We used DAPI for nuclear staining and Diamond antifade medium (Life Technologies) for mounting the slides.
Sample scanning, spectral unmixing, and quantification of signals were conducted with the Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya), using the Phenochart and InForm 2.4 software (Akoya). The number of positive cells belonging to each specific phenotype was given as number of cells/square micron.

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.