After staining, tissue section slides were scanned with Vectra 2 (Akoya, Boston, MA, USA) at 4× magnification, and then 10 fields of view were randomly selected per slide with PhenoChart software (Akoya, Boston, MA, USA) to be scanned with Vectra 2 at 20× magnification and analyzed with the InForm v.2.4 software (Akoya, Boston, MA, USA). A spectral library algorithm was created to unmix each individual signal, and the following pseudocolors were applied for image analysis: CD4 (yellow), CD68 (red), CD8 (green), CD20 (pink), and PCK (magenta). The InForm software was used to segment tissue compartments (epithelium vs. stroma) and subcellular compartments (nucleus, membrane, and cytoplasm). Individual cell segmentation was performed, and cell phenotypes were quantitated as cell density (cells/mm2) (Figure 1A).
Shapiro D.D., Lozar T., Cheng L., Xie E., Laklouk I., Lee M.H., Huang W., Jarrard D.F., Allen G.O., Hu R., Kinoshita T., Esbona K., Lambert P.F., Capitini C.M., Kendziorski C, & Abel E.J. (2024). Non-Metastatic Clear Cell Renal Cell Carcinoma Immune Cell Infiltration Heterogeneity and Prognostic Ability in Patients Following Surgery. Cancers, 16(3), 478.
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