Clone 2g9

Skin from the back of the mice was harvested and froZEN directly in OCT. 8 μm sections were taken, fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at RT and permeabilized with 0.1% TritonX-100 for 15 min. Skin sections were blocked before staining with 2.5% Normal Goat Serum (Sigma-Aldrich) and 1% BSA for 1 h at RT. Staining was performed with anti-MHCII (1:200 dilution, clone 2G9, BD Pharmingen, San Diego, CA, cat. 553623) and anti-Langerin (1:50 dilution, clone eBioL31, eBioscience cat. 12–2075–82) overnight at 4 °C. Sections were mounted using Pro-Long^TM Gold antifade reagent with DAPI (Invitrogen). Images were obtained with a Zeiss LSM780 confocal microscope and analyzed with ImageJ and ZEN (Zeiss) software.

For functional analysis of pDCs (Ifn-α production and costimulatory molecule upregulation), bones (femur, tibia, collarbone) of Apoe^-/- control and Apoe^-/-BDCA2-DTR mice were harvested and flushed with Hank’s medium (Hanks’ Balanced Salt Solution + 0.3 mmol/l EDTA + 0.1% BSA). Cell suspensions were treated and stained according to FACS protocol with following antibody cocktail: anti-CD45 (eBioscience, clone 30-F11), anti-B220 (eBioscience, clone RA3-6B2) and anti-SiglecH (eBioscience, clone eBio440c). After cell sorting, sorted pDCs were cultured in 24 well flat-bottom plates (1x10⁵ cells/well) in RPMI1640 Medium with L-Glutamine (Gibco by life technologies) and 1% Penicillin/Streptavidin with or without 5 μg/ml CpG oligodeoxynucleotides (ODN 1585, InvivoGen). After incubating pDCs 12 hours at 37°C, cell supernatants were used for Ifn-α ELISA and costimulatory molecule upregulation was measured with FACS using anti-CD86 (eBioscience, clone GL1) and anti-MHC class II (BD Pharmingen, clone 2G9) antibodies. To analyze the expression of DTR on pDCs, splenic pDCs of Apoe^-/- control and Apoe^-/-BDCA2-DTR mice were stained and measured according to the FACS protocol with an anti-hDTR antibody (human HB-EGF, clone #125923, R&D Systems).

Antibodies used in these studies included: anti-CD8a, clone 53–6.7; anti-CD11b, clone M1/70; anti- CD11c, clone N418; anti-CD103, clone 2E7; anti-I-A/I-E (MHC Class II) for flow cytometry and epidermal sections, clone M5/114.15.2 (all from Biolegend, San Diego, CA); anti-I-A/I-E for small intestine cryosections, clone 2G9 (BD Biosciences, Franklin Lakes, MJ); and anti-CD207, clone 929F3.01 (Novus Biologicals, Littleton, CA).