Alexa 568 conjugated goat anti rabbit igg

LPS (Lot. No. L8274) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse CD11b (M1/70) and MHC Class I (H2K^d, SF1-1.1) monoclonal (m) Abs were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse CD16/CD32 (93), and Gr-1 (RB6-8C5) mAbs were purchased from eBioscience (San Diego, CA, USA). Anti-CD11c (N418), CD80 (16-10A1), MHC Class II (I-A/I-E, M5/114.15.2) and 7-amino-actinomycin D (7AAD) from Tonbo Biosciences (San Diego, CA, USA). Rabbit anti-MRC1 polyclonal (p) Ab was from Bioss Antibodies (Woburn, MA, USA). Alexa 568-conjugated goat anti-rabbit IgG was from Molecular Probes (Eugene, OR, USA).

HUVECs were treated with/without 2 µM of rMASP-1, in 100 µl Comp-AIM-V for 25 minutes, fixed and stained with rabbit-anti-human phospho-CREB (P-CREB, 1∶200, Cell Signaling Technology Inc.) antibody followed by Alexa568 conjugated goat-anti-rabbit IgG (1∶500) and Hoechst 33342 (1∶50000, Molecular Probes/Invitrogen) as described previously [8] (link). All analyses were performed using the original, unmodified images; for visualization, the pictures were modified according to a standardized procedure, using Adobe Photoshop CS, without gamma-correction.

Frozen tissue sections were incubated with a monoclonal mouse anti-BrdU antibody (Lab Vision, Fremont, Calif.). After blocking in Envision blocking buffer, sections were placed in primary antibody overnight at 4°C to 8°C. On the following day, the sections were incubated with Alexa 568 (Molecular Probes, Eugene, OR.) conjugated goat anti-mouse IgG. Endothelial-like cells were stained for von Willebrand factor (vWF) (primary antibody: polyclonal rabbit anti-vWF antibody, H-300, Santa Cruz, Dallas, Texas; secondary antibody: Alexa 568 conjugated goat anti-rabbit IgG, Molecular Probes). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). For colocalization study, BrdU-positive cells were identified (primary antibody: monoclonal mouse anti-BrdU antibody, Lab Vision; secondary antibody: Alexa 488 conjugated goat anti-mouse IgG, Molecular Probes).

Immunohistofluorescence assay was performed according to the method described before [19 (link)]. Anti-α-SMA antibody and anti-F4/80 (Abcam, UK) were used. All of these antibodies were used at a dilution of 1/100. Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) and Alexa 568-conjugated goat anti-rabbit IgG (Invitrogen) were used as secondary antibodies.

Cells grown on 2% gelatin (Sigma–Aldrich) coated coverslips (Carolina Biological Supply Company, Burlington, NC) were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.2% Triton X-100 in 1× PBS (pH 7.4). After pre-blocking for 1 h at room temperature with 1% normal goat serum/1× PBS, cells were incubated overnight at 4°C in a humidified chamber with the primary mouse anti-NEP (1:100, Abcam) or rabbit anti-HNE (1:200, Millipore) antibodies. At the end of the incubation period, the cells were rinsed three times with 1× PBS containing 0.05% Tween-20 (PBS-T) and then incubated with the secondary Alexa 488-conjugated goat anti-mouse or Alexa 568-conjugated goat anti-rabbit IgG (1:500, Invitrogen) for 60 min at room temperature. All primary and secondary antibodies were diluted in PBS-T with 2% normal goat serum. After rinsing with 1× PBS, the coverslips were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen), and viewed and photographed on a Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Immunofluorescence staining was repeated at least three times.