Fluoromount g mounting medium with dapi

Preparation and staining of transiently transfected HEK-293 cells were the same as described above for flow cytometry. Cells were stained with 1C7 antibody and as secondary antibody 1 μl of PE goat anti-mouse IgG (Biolegend) to detect cell surface-expressed KIR3DS05 and were subsequently fixed, permeabilized, blocked and stained intracellularly with 0.5 μg anti-myc antibody (Abcam) and 0.25 μg APC conjugated goat anti-rabbit IgG (Abcam) to detect myc-tagged adaptors. Finally, a drop of Fluoromount-G mounting medium with DAPI (Thermo Fisher Scientific) was applied on a microscope glass slide (Carl Roth), and the stained cells were transferred onto the mounting medium and incubated for 5 min at RT, before fixing the cells with a cover slip (24 × 24 mm). The images were captured with the Plan-Apochromat 63x/1.40 oil objective in confocal laser microscope LSM 800 (Carl Zeiss) fitted with ZEN 2.3 software (Carl Zeiss) and mean fluorescence intensity (MFI) value of KIR3DS05 (1C7) and FcRγ/DAP12 (anti-myc) in 11 individual stained cells were measured using the same software.

Human HEK293 cells (obtained from ATCC, catalog number: CRL-1573) were cultured at 37 °C and 5% CO₂ in DMEM (ThermoFisher Scientific), supplemented with 10% fetal bovine serum (ThermoFisher Scientific), 100 units/mL penicillin and 100 µg/mL streptomycin (ThermoFisher Scientific). For transient transfection, 50,000 cells were seeded per well (24-well plate, 500 µl growth medium). After 24 h, cells were transfected with Lipofectamine 3000 (ThermoFisher Scientific) according to the manufacturer’s instruction using 3 µl transfection reagent per µg DNA and 2 µl of p3000 reagent per µg DNA. Per well 500 ng of DNA was used. After 24 h, cells were fixed in 3.7% formaldehyde solution and mounted using Fluoromount-G Mounting Medium, with DAPI (ThermoFisher Scientific).

Lesional AD skin biopsies were cryo-sectioned to 6 µm thickness, fixed with 4% PFA, and blocked with a 2x Casein solution (B6429) (Sigma-Aldrich, St. Louis, MO, USA). Slides were consecutively incubated anti-S1P (1:200) (Z-P300) (Echelon Biosciences, Salt Lake City, UT, USA), goat anti-mouse Alexa Fluor 488 secondary antibody (1:2000) (15607878) (ThermoFisher Scientific, Waltham, MA, USA) and the basophil specific anti-2D7 Alexa Fluor 647 (RRID: AB_1967142) (1:100) (BioLegend, San Diego, CA, USA). Slides stained with the respective isotypes or only the secondary antibody were utilized as experimental controls. Slides were subsequently mounted with Fluoromount-G Mounting Medium with DAPI (00-4959-52) (ThermoFisher Scientific, Waltham, MA, USA) and analyzed with the Olympus BX63 fluorescence microscope and the CellSens image analysis software (Olympus, Tokyo, Japan).

For immunofluorescence, macrophages were cultivated on cover slides (VWR, Germany). Cells were washed with PBS (Carl Roth, Germany) and fixed with methanol (VWR, Germany) for 10 min at room temperature (RT). Cover slides were then incubated for 30 min with blocking solution (PBS + 0.1% bovine serum albumin, VWR) at RT. Afterwards, cells were incubated with monoclonal mouse anti-human VISTA (R&D Systems, USA) for 1 h at RT. After washing, cells were stained with secondary goat anti-mouse IgG Alexa Fluor 594 (Thermo Scientific, USA), in the dark at RT. Cover slides were mounted with Fluoromount-G Mounting Medium, with DAPI (Thermo Scientific, USA). Image capture was performed using an Olympus BX63 (Olympus, Japan) fluorescence microscope. Images were processed by Olympus cellSense and ImageJ software.

Paraffin-embedded tissue sections (5 µm) were dewaxed in xylene and rehydrated. Antigen retrieval was performed at pH 6 before blocking in PBS containing 5% normal donkey serum, 0.1% BSA and 0.3% Triton for 30 minutes. Sections were then incubated simultaneously overnight at 4°C with primary polyclonal goat anti-CD31 antibody (1:100) and primary polyclonal rabbit anti-3-nitrotyrosine (3-NT) antibody (1:1000). After washing in PBS containing 0.3% Triton, sections were incubated 1h at room temperature with secondary antibodies (anti-goat Alexa 555 and anti-rabbit Alexa 647, 1:300) and washed. Coverslips were mounted with Fluoromount-G™ Mounting Medium, with DAPI (ThermoFisher). Negative control sections were incubated only with the secondary antibodies to set the fluorescence threshold. Slides were scanned with NanoZoomer S60 slide-scanner. Fiji (ImageJ, version 1.52h) was used to quantify the percentage of 3-NT area present into CD31 area. The Fiji script used for the analyses is given in the Supplementary information (Supplementary File S2). The immunofluorescence analyses and quantification were performed in a blinded manner by a single investigator (AD).