Fig 1: Development of a multispectral panel for the topological examination of DCs. (A) Representative images showing staining for CLEC9A (yellow), CD20 (cyan), CD11c (orange), CD123 (red), CD8 (pink), CD4 (green), Ki67 (magenta) and JOPRO-1 (blue) in a tonsillar tissue section and the distribution of each protein marker with respect to the MZ, LZ and DZ (middle panels). Red dotted square enclosures in lower middle panels denote the areas that are presented magnified in the lower middle panels. (B) Gating strategy used for the sequential positional immunophenotyping of CD4, CD8, CD20, CD11c+, CD11c+CLEC9A+CD123- and CD11c-CD123+CLEC9A- in GC (CD20dimKi67hi), F (CD20hi/dim) and EF (CD20lo) areas using histocytometry. (C) Box graphs and box plots showing the relative frequencies of CD11c+, CD11c+CLEC9A+CD123- and CD11c-CD123+CLEC9A- immune cells in tonsillar tissue as well as in CD20hi/dim areas (upper panels) and relative frequencies of CD20, CD4, CD8 and CD11c+ immune cells in nine individual follicles. Images were acquired at 40x (NA 1.3) with 1% magnification. Scale bars are 100um for all panels displaying an entire follicle), 20um in the middle panel close ups and 5um in the right panel single staining close ups.