Scanning device

Cells were seeded in appropriate medium on cover slips in 12-well plates (BD Falcon, Le Pont-de-Claix, France). Once adherent, cells were fixed (formaldehyde, 4% in PBS, at RT, 15 min) permeabilized with PBS-0.05% saponin (20 min at RT) and saturated (BSA, 4% in PBS, 30 min). The cells were then successively incubated with primary antibodies to PRAAHG or to PAVIRF peptides, or with SAB L194 antibody, for 120 min at RT and secondary AlexaFluor488-labeled antibodies to rabbit IgG for 60 min. The cell nuclei were labeled with 1 μM Draq5 for 30 min. Confocal microscopy acquisitions were performed using a Leica SP5 microscope coupled with a Leica scanning device (Leica Microsystems, Mannheim, Germany). Images were recorded with LAS AF Lite acquisition software and were analysed with the public-domain ImageJ software (NIH; http://rsb.info.nih.gov/nih-image/).

Frozen tissue sections were subjected to immunodetection after saturation with PBS 4% BSA. Sections were incubated with primary antibodies diluted in PBS 1% BSA. Gr-1⁺ and Foxp3⁺ and TCRγδ⁺ cells were detected by incubation with the rat anti-Gr-1 (BD Pharmingen), the rabbit anti-Foxp3 (Abcam), and the armenian hamster anti-TCRγδ (Biolegend) antibodies, respectively. After three washes in PBS, samples were incubated for 1 h with either Alexa Fluor 488-, Alexa Fluor 594- (life technologies) or Cy3-conjugated goat (Jackson Immunoresearch) immunoglobulin [Ig], raised against rat, rabbit and armenian hamster Igs, respectively, then washed in PBS. Nuclei were labeled with Draq5 and sections were mounted in ProLong Gold (Invitrogen). Confocal microscopy acquisitions were performed using a Leica SP5 microscope coupled with a Leica scanning device (Leica Microsystems, Mannheim, Germany). Images were recorded with LAS AF Lite acquisition software and were analyzed with the publicdomain ImageJ software (NIH; http://rsb.info.nih.gov/nih-image/).