Cd209

Six-micron frozen DLE (n = 6–8) and normal (n = 4–5) skin sections fixed in acetone were blocked in 5 % normal goat serum in PBS with 0.3 % Triton X-100 for 1 hour. Slides were then incubated with rabbit anti-human primary antibodies—CD127 (Abcam, Cambridge, MA, USA), CD209 (Abcam), CXCL10 (PeproTech, Rocky Hill, NJ, USA), tumor necrosis factor-α (TNF-α) (Novus, Littleton, CO, USA), and TGF-β (Novus)—overnight followed by PBS washes. Alexa Fluor^® 488-conjugated goat anti-rabbit antibodies (Life Technologies, Carlsbad, CA, USA) were added for 30 minutes followed by PBS washes, except for TGF-β and TNF-α antibody-stained slides, whose signals were amplified by using the TSA Biotin kit in accordance with the instructions of the manufacturer (PerkinElmer, Waltham, MA, USA). Mouse anti-human CD163 antibody (Novus) or mouse anti-human CD3 antibody (eBioscience, Inc., San Diego, CA, USA) was added for 1 hour, followed by PBS washes. Slides were incubated with Alexa Fluor^® 594-conjugated goat anti-mouse antibodies (Life Technologies) for 30 minutes and washed with PBS. Slides were coverslipped with Vectashield mounting medium and analyzed. Images were acquired with an Olympus BX60 fluorescent microscope (Olympus, Tokyo, Japan).

Tumor hypoxia was studied by intraperitoneal injection of 60 mg/kg pimonidazole (PIMO) hydrochloride (Hypoxyprobe™-1 Kit, Chemicon) 1 h before tumor harvest. Cryostat sections were fixed and permeated. PIMO, F4/80, CD209 and CD31 (Abcam, Cambridge) antibodies were used, followed by Alexa Fluor 488 or 594. Nuclei were visualized by staining DAPI.