Cd207

Skin specimens were embedded in OCT and frozen. 5-µm sections were cut using the Leica CM 1950. For immunofluorescent staining, tissue sections were fixed in 4% PFA for 10 min at room temperature and washed with PBS containing 3% BSA and 10% saponin. Sections were quenched with 0.5 M Glycine for 5 min, washed, and blocked with PBS/BSA/Sapo for 30 min at room temperature. Tissue sections were incubated overnight at 4°C with either polyclonal rabbit anti–human antibodies, PSME1 (2.5 µg/ml; Novus Biologicals), Sec61a (0.5 µg/ml; Pierce Antibodies), TAP2 (2 µg/ml; LifeSpan BioScience) or an isotype control. All sections were stained for mouse anti–human Langerin (CD207; 2 µg/ml; Beckman Coulter) overnight at 4°C. Sections were then washed and incubated with anti–mouse Alexa Fluor 488 (1/200) and donkey anti–rabbit Alexa Fluor 647 (0.6 µg/ml; Jackson ImmunoResearch Laboratories) for 2 h followed by 4’,6’-diamidino-2-phénylindole (DAPI) for 10 min at room temperature. Sections were then washed in PBS and mounted (Invitrogen). Images were acquired using an Olympus Confocal Microscope FV1000 using Fluoview software. Image analysis was performed using ImageJ software.