Goat anti human il 6

Carotid artery samples were collected immediately, frozen in liquid nitrogen and stored in -75°C. Air-dried serial frozen sections were fixed in ice-cold acetone and exposed to the following primary antibodies: monoclonal mouse anti-human CD68, clone PG-M1 (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, USA), goat anti-human IL-6 (1:100, R & D Systems, Inc, USA) and monoclonal mouse Anti-C-Reactive Protein, clone CRP-8 (1:100, Sigma-Aldrich, USA), The immuno-reactions were visualized using the DAKO LSAB method and DAKO LSAB + Kit, HRP (DAKO, Denmark) [21 (link)]. Controls without primary antibodies were run for each protocol, resulting in consistently negative results. Isotype controls were tested with normal serum from the same animal or species as the primary antibody or the same immunoglobulin isotype. The slides were washed, mounted with an aqueous mounting medium and photographed within a few hours under a digital microscope camera (Olympus Upright Light Microscope). Immunohistochemical quantification for CRP and IL6 were assessed by image processing software “Image Pro Plus version 6”. Light intensity and contrast were standardized for a respective sections with an appropriate control. For analysis of CRP and IL6 staining three 200 μM × 200 μM areas where chosen by blinded analysis from each region and reported as an average of area IOD.

Human monocytes derived macrophages (HMDM) plated on a round caver glass at 16-mm dish were fixed in the absolute methanol for 10 min. After rinsing with cold PBS (pH 7.4) cells were permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. After blocking, antihuman-CRP antibody clone CRP-8 (Sigma-Aldrich, USA) and goat anti-human IL-6 (1:00, R & D Systems, Inc, USA) were respectively added and incubated at room temperature for 2 hours followed by incubation with CyTM3 conjugated Donkey Anti-mouse IgG (Jackson Immuno Research Labs, Baltimore, USA) and FITC conjugated Rabbit Anti-Goat IgG (Jackson Immuno Research Labs, Baltimore, USA) for 1 hour. Following removal of the antibodies, and nucleus counterstaining with To-pro-3 iodide (Invitrogen, Carlsbad, Ca, USA). Cells were rinsed with PBS, cover glass with cells removed from dish and mounted with UltraMount (Lab Vision, UK) [22 ]. All control samples were processed without primary antibody. Fluorescence was immediately observed using either an Axioscop 2 or Leica laser scanning confocal microscope (Bensheim, Germany) with image processing software “Image Pro Plus version 6”. Light intensity and contrast were standardized for respective cells samples with an appropriate control.

Human NHOst cells were grown to confluence in 4-well chamber slides (Sarstedt, Numbrecht, Germany), fixed for 10 min at room temperature with 4% paraformaldehyde (EMD Biosciences), then circled with an ImmEdge Hydrophobic Barrier Pen (Vector Laboratories). Next, cells were permeabilized for 10 min using 0.2% Triton-X (Sigma) in PBS. Non-specific binding was blocked with Dako Universal Blocking Buffer (Dako Products) for 1 h at room temperature. The slides were incubated overnight at 4 °C with either mouse anti-human osteocalcin (R&D Systems, 10 μg/ml), rabbit anti-human RUNX2 (Abcam, 1:500), mouse anti-human alpha-smooth muscle actin (1:100, Abcam), rabbit anti-human alkaline phosphatase (1:100, Abcam), rabbit anti-human osteopontin (1:500, Abcam), or goat anti-human IL-6 (40 μg/mL, R&D Systems), Next, slides were incubated for 1 h at room temperature with either donkey anti-mouse 594, chicken anti-rabbit 594, donkey anti-goat 488, or goat anti-rabbit 488 (1:1000, Biotium). Cells were stained with DAPI (0.2 ng/μl) then mounted with Fluoromount G (Southern Biotech). The cells without the primary antibody served as negative controls. Images were viewed using an Olympus FV 3000 fluorescent microscope (Olympus) equipped with Hamamatsu color camera (Hamamatsu Photonics).