Fluoview fv10i confocal microscope system

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.

mGCs were plated on 18 mm microcover glasses (Matsunami, Osaka, Japan) for 24 h and subsequently fixed with 4 % paraformaldehyde in PBS for 30 min at room temperature. The cells were then washed with PBS and permeabilized with 0.2 % Triton X-100 in PBS for 15 min at room temperature. After being blocked with 1 % BSA in PBS, cells were stained for FSHR by incubation with a 1:100 dilution of an anti-FSHR polyclonal antibody (Bioworld Technology, St. Louis Park, MN, USA), followed by incubation with a 1:200 dilution of an Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Life Technologies, Carlsbad, CA, USA) for 1 h. PBS was used as negative controls for primary and secondary antibodies to exclude nonspecific staining. Nuclei were stained with a 1:5000 dilution of DAPI (Vector Laboratories, Burlingame, CA, USA). Images were visualized using a FLUOVIEW FV10i confocal microscope system (Olympus, Tokyo, Japan).

For immunofluorescence, cultured HDMECs were grown on collagen-coated cover slips. Cells were treated with OSM and IL-6+sIL6R for 48 h and 72 h, or siRNA for ERG and FLI1. Cells were fixed with 4% paraformaldehyde for 15 min. Non-specific protein binding was blocked with 3% BSA for 1 h. Next, cells were incubated at 4 °C overnight with primary antibody. After washing, cell cultures were incubated with appropriate fluorophore-conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1.5 h. Skin biopsies were embedded in OCT and fixed in acetone:methanol (1:1). Sections were blocked in 3% BSA for 1 h, before the addition of primary antibodies diluted in 1% BSA. After washing, sections were incubated in appropriate fluorophore-conjugated secondary antibodies for 45 min. Cells and biopsy sections were mounted on slides using Vectashield with DAPI (Vector Laboratories, Burlingame, CA) and examined using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA) at 488 nm (green), 594 nm (red), and 405 nm (blue). Secondary Alexafluor antibodies (Invitrogen, Carlsbad, CA) were used for each stain. Primary antibodies and concentrations are listed in Supplemental Table II.

For all immunofluorescence staining tumor samples were directly embedded in O.C.T. compound, flash frozen, and stored at -80°C. Staining was performed on 5 μm cryosections of tumor. Briefly, slides were blocked with a blocking solution (3% BSA [Sigma-Aldrich], and 0.1% Triton^® X-100 in PBS) for 2 h. After washing, tissue sections were incubated at 4°C overnight with primary antibodies: rabbit polyclonal PDGFRB (Cell Signaling, Danvers, MA), mouse anti-E-Cadherin (BD Biosciences) and goat polyclonal VE-Cadherin (Santa Cruz, CA). After washing, tissue sections were incubated with secondary Ab: Alexa fluor 488 donkey anti-rabbit (Invitrogen, Grand Island, NY), Alexa fluor 594 donkey anti-mouse, and Alexa fluor 594 donkey anti-goat (Invitrogen, Grand Island, NY) for 1.5 h. Coverslips were mounted on slides using Vectashield with DAPI (Vector Laboratories, Burlingame, CA) and examined using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA) at 488 nm (green), 594 nm (red) and 405 nm (blue).