Gl7 biotin

OCT-embedded lymph nodes were sectioned at 7 microns onto positively charged glass slides and stored at −80 °C until staining. The slides were fixed for 10 min in 75:25 acetone/ethanol at room temperature. Sections were serum blocked for 30 min at room temperature with PBS containing 10% v/v normal mouse serum, 10% v/v normal rat serum, 10% v/v normal donkey serum, 2% w/v bovine serum albumin, 0.05% w/v sodium azide, and 1 μg/ml anti-CD16/32 clone 2.4G2 (clone 2.4G2 ATCC HB-197). Biotin blocking was performed with an Endogenous Biotin Blocking Kit (ThermoFisher E21390) per the manufacturer’s instructions. Slides were then stained with a primary antibody cocktail of anti-CD90.2-FITC (1:100, clone 30-H12, Biolegend, 105306), GL7-Biotin (1:20, eBioscience, 13-5902-81), and anti-IgD-A647 (1:50, clone 11–26c.2a, Biolegend, 405708) in serum blocking buffer. Secondary stain consisted of anti-FITC-A488 (1:100, polyclonal, Life Technologies, A11090) and Strepavidin-Hilyte555 (1:20, Anaspec, 60666) in serum blocking buffer. Coverslips were mounted with Pro-Long Gold Antifade Mountant (ThermoFisher, P36930). Between each step, slides were washed with PBS 4× 30 s and dried on the grate of a BSC. Slides were cured overnight and imaged on a Zeiss Axio Observer A1 using Zen software (v2.0.0.0).

Antibodies specific for: CD-8a-488 (clone 53-6.7 Biolegend), IL-2-488 (clone JES6-5H4 eBioscience), CD169-488 (clones SER4 and MOMA-1 in house), CD11a-FITC (clone M17/4 eBioscience), CD11c (clone N418 eBioscience), CD44-FITC (clone HI44a ImmunoTools), CD11c-PE (clone N418 eBioscience), CD38-PE (clone 90 eBioscience), CD4-PE (clone GK1.5 eBioscience), CD8a-PE (clone 53-6.7 eBioscience), GL7-biotin (eBioscience), B220-ef450 (clone 6B2 eBioscience), KLRG1-ef450 (clone 2F1 eBioscience), CD127/IL7Rα-APC (clone A7R34 Biolegend), CD8a-APC (clone 53-6.7 eBioscience), IFNγ-APC (clone xM61.2 eBioscience), CD62L-PECy7 (clone MEL-14 Biolegend), CD8a-PECy7 (clone 53-6.7 eBioscience), CD4-PERCPCy5.5 (clone RM4-5 eBioscience), CD8a-PERCPCy5.5 (clone 53-6.7 Biolegend). OVA-488 (Invitrogen). H-2Kb-SIINFEKL Tetramers (LUMC, Leiden). LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen) was used according to manufacturers' protocol.