Fig. 1: Visualization of infiltrating immune cells in follicular thyroid carcinoma (FTC) with minimal capsular invasion and adjacent normal tissue via 12-color multiplex immunohistochemistry (mIHC).

A, B Formalin-fixed paraffin-embedded (FFPE) FTC tissue sections (n = 13) were subjected to immune detection using a panel of 14 lineage-selective antibodies to reveal the complex distribution and density of nine immune cell lineages (Supporting Information, and Supplementary Tables 1 and 2). Pseudo-colored merged composite images are shown. Boxed region (left images) indicates areas shown at higher magnification to the right. Antibodies and color annotations are shown at the bottom. Scale bar of left images = 500 μm. Scale bar of right images = 100 μm. C Whole hematoxylin and eosin (HE)-stained image of FTC tissue section is shown in the upper left panel. Scale bar = 5 mm. Intratumoral region was divided into tumor center (TC), subcapsular region (SC), and capsular invasion (CI), and extratumoral region was divided into adjacent stroma of CI (CI-AS), peritumoral stroma (PS), and adjacent normal (AN). The details of tissue segmentation are described in supplementary methods. The left panel shows the magnified HE-stained images and the right panel shows the corresponding mIHC images of hematoxylin and CD45 staining. Scale bar = 500 μm. D CD45 immunostaining intensity in FTC sections is presented by a 3D heatmap designed using ImageJ/Fiji Version 1.51 s.