Tonsils were frozen in Tissue-Tek O.C.T™ Compound (Sakura Finetek). Multiplex microscopy analyses were done on a 7μm-section tissue using Akoya Phenocycler platform. Tissue was stained following Akoya’s recommendation for staining using a panel described in Table S2 . Computational analysis is detailed in Supplementary Materials .
6μm-sections were prepared and mounted on SuperFrost™ Plus glass slides (Fisher Scientific), fixed in cold acetone for 20 min and air dried for standard confocal microscopy analyses. Non-specific antibody binding was avoided using Blocking Reagent™ (Perkin Elmer) for 30 min. Sections were then incubated overnight with anti-human CD38 (EPR4106, abcam), FOXP3-eFluor570 (236A/E7, Invitrogen) BCL6-AF647 (K112–91, BD Bioscience) antibodies at 4 °C overnight. To detected CD38 protein, sections were incubated with anti-rabbit AF488 (Invitrogen) antibodies for 2h at room temperature. After paraformaldehyde 4% fixation (Electron Microscopy Sciences) for 20 min, slides were stained with DAPI (1 μg/ml, Sigma) for 15 min at room temperature and mounted with fluorescent mounting medium (DAKO). Images were acquired using a Leica TCS SP8 Confocal and analyzed with ImageJ software.
6μm-sections were prepared and mounted on SuperFrost™ Plus glass slides (Fisher Scientific), fixed in cold acetone for 20 min and air dried for standard confocal microscopy analyses. Non-specific antibody binding was avoided using Blocking Reagent™ (Perkin Elmer) for 30 min. Sections were then incubated overnight with anti-human CD38 (EPR4106, abcam), FOXP3-eFluor570 (236A/E7, Invitrogen) BCL6-AF647 (K112–91, BD Bioscience) antibodies at 4 °C overnight. To detected CD38 protein, sections were incubated with anti-rabbit AF488 (Invitrogen) antibodies for 2h at room temperature. After paraformaldehyde 4% fixation (Electron Microscopy Sciences) for 20 min, slides were stained with DAPI (1 μg/ml, Sigma) for 15 min at room temperature and mounted with fluorescent mounting medium (DAKO). Images were acquired using a Leica TCS SP8 Confocal and analyzed with ImageJ software.