Immunofluorescence and Immunohistochemical Staining of Monocytes and Skin Tissues

Monocytes were stained with mouse monoclonal antibodies against CD14-PE (BD Pharmingen), BFRF1 [18 (link)], gp-350-220 [13 (link)], rabbit TLR8 (Cell Signaling), and secondary-antibodies-Cy3-conjugated (Jackson IR, West Grove, PA, USA), Alexafluor-350 goat-anti mouse antibody (Invitrogen, Grand Island, NY, USA), or 488-labeling (Zenon kit; Invitrogen). Coverslips were mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) and immunofluorescence staining was examined using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA, USA) at 488 nm (green), 594 nm (red), and 405 nm (blue). Paraffin sections of skin tissues were stained using mouse mAb against CD163 (AbD Serotec, Raleigh, NC, USA) and Zta/Zebra (Argene, bioMérieux, Inc., Durham, NC, USA) as previously described [13 (link)]. Immunohistochemical staining was examined using the Olympus BH2 microscope.