Journal: iScience
Article Title: Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches
doi: 10.1016/j.isci.2023.107261
Figure Lengend Snippet: Compartmentalization of tonsillar cell types and soluble mediators revealed by multiplex imaging analysis (A) Representative hematoxylin and eosin (H&E) stained human tonsil section. Relevant anatomical segments are labeled for tonsillar crypt (C), germinal center (GC), mantle zone (MZ), crypt epithelium (E), T cell zone (TZ) and extrafollicular area (EF). (B) Representative multiplex confocal images showing the distribution of B, CD4 and CD8 cell subsets in a follicular area and adjacent ‘T cell area’. Zoomed areas (white boxes) are also shown for the visualization of the expression of relevant biomarkers on individual cells. Scale bars top row are 100 μm. (C) Bar graphs showing the relative expression (as frequency of the corresponding parental population) of Ki67 + B cells and PD-1 + CD57 + CD4 T cells as well as the frequency (of total cells) of CD8 and CD68 subsets in different tonsillar areas (EF = extrafollicular, MZ = mantle zone, GC = germinal center) (upper panel), Kruskal-Wallis (± SEM) statistic test used significant differences notated. The correlation between Ki67 + GC B cells and PD-1 + CD57 + CD4 T FH cells is shown (lower panel), Simple linear regression statistical test used. (D) Representative multispectral images showing the distribution of FDCs (green), CD14 (yellow), CXCL13 (red) and IL17 (cyan) with respect to the follicular areas (CD20, blue) in one tonsil. Inserts are showing the expression of IL17 in zoomed areas (white circles). Scale bars are 200 μm.
Article Snippet: FFPE tissue sections of 8 μm were prepared using a microtome (Leica) and processed (heat drying, deparaffinization) for antigen retrieval (Borg RTU, Biocare Medical) and antibody staining using titrated antibodies as recently described., The following combination of antibodies was used: IgD (primary rabbit, clone EPR6146, abcam), CD8 (primary mouse IgG2b, clone 4B11, Invitrogen), CD68 (primary mouse IgG1, clone KP-1, DAKO), CD20-eF615 (clone L26, eBioscience), Ki67-AF647 (clone B56, BD Horizon), CD4-AF700 (goat polyclonal, R&D), PD-1-AF488 (goat polyclonal, R&D), CD57-BV480 (clone NK1, BD Horizon), and JoJo (nuclear marker, Life Technologies).
Techniques: Multiplex Assay, Imaging, Staining, Labeling, Expressing
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