Tissuequest quantitation software

Images of the tissue sections were acquired using the TissueFAXS software (TissueGnostics) connected with a Zeiss Axio Observer Z1 inverted microscope (Olympus). The quantitative analysis of the cells of the different phenotypes within the images was done using the TissueQuest quantitation software (TissueGnostics).

Multiplex fluorescent immunohistochemistry experiment was performed using the Opal™ 4-Color Manual IHC kit (PerkinElmer) according to the manufacturer’s instructions. Briefly, tonsil tissue samples fixed with 4% formalin for a minimum of 48 hours were paraffin-embedded. Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), GZMB (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals). Opal fluorophores: FITC (Opal520) was used for p24 and CD103; Texas-Red (Opal570) was used for CD8 and CD4; then Cy5 (Opal690) was used for CD8, CXCR5, Granzyme-B and CD69 signal generation in the different IHC experiments performed. DAPI was used as the nuclear counterstain. Images were acquired on a Zeiss Axio Observer Z1 inverted microscope (Olympus) and analysed with TissueFAXS imaging and TissueQuest quantitation software (TissueGnostics).