Dabco antifade reagent

5 × 10⁵ cells were fixed in suspension in 1% paraformaldehyde and 0.0075% glutaraldehyde and washed three times in PBSAG before being cytospun onto coated coverslips. Cells were then permeabilized with 0.05% Triton X-100 for 5 min at room temperature and then blocked in PBS-4% BSA for 45 min, incubated with primary antibodies in PBS-4% BSA for 1 h, washed with PBS, and incubated for 1 h with the appropriate secondary antibodies. Coverslips were washed and mounted on microscope slides using Mowiol (Calbiochem, San Diego, USA) containing 2.5% (w/v) DABCO antifade reagent (Sigma-Aldrich). Confocal images were taken using a Leica SPE single-channel confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope.

LSEC were grown on collagen-coated coverslips and loaded with 0,1 mg/ml OVA (for coculture with OT-1 T cells) or left untreated (for coculture with DesTCR T cells) for 1–2 hours. To ensure that the start of LSEC/T cell interaction was synchronized naïve T cells were centrifuged onto the LSEC for 1′ at 1000 rpm. For TIRF microscopy cells were fixed after the indicated time-points in 4% paraformaldehyde, blocked with Tris-Buffered Saline containing 1% BSA/1% donkey serum (Jackson Immunoreasearch) and stained with anti-TCRβ (H57-597, eBioscience), Alexafluor-488 Goat-anti-Hamster IgG (H+L, Molecular Probes) as secondary antibodies or anti-CD11a (I21/7, Southern Biotech) antibodies, Alexafluor-488 Goat-anti-Rat IgG (H+L, Molecular Probes) as secondary antibodies. After washing coverslips were mounted in ProlongGold (Invitrogen), supplemented with 50 µg/mL DABCO anti-fade reagent (Sigma-Aldrich), and analyzed. For confocal microscopy cells were incubated with avidin/biotin blocking agent (Invitrogen) and stained with biotinylated anti-TCRβ and unlabeled anti-CD11a antibodies and Cy5-labeled streptavidin and Cy3-labeled anti-Rat-IgG (Jackson Immunoresearch).