Fitc conjugated anti mouse igg

Antibodies used were as follows with dilutions in parentheses. Rabbit anti-eIF4G (1:10,000), anti-eIF4E (1:3000) and anti-eIF4A (1:2000) were as described previously (Coldwell et al., 2012 (link); Morley and Pain, 1995 (link)). Mouse monoclonal anti-eIF4G (sc-373892; 1:3000 for western blotting, 1:50 for immunofluorescence), rabbit polyclonal anti-SUMO1 antibody (sc-9060; 1:2500 for western blotting, 1:50 for immunofluorescence), mouse monoclonal anti-SUMO1 (sc-5308; 1:2500 for western blotting, 1:50 for immunofluorescence), rabbit polyclonal antibody against both SUMO2 and SUMO3 (sc-32873; 1:2500) were from Santa Cruz Biotechnology. Mouse monoclonal anti-FLAG (F1804; 1:1000 for western blotting, 1:400 for immunofluorescence) was from Sigma-Aldrich. Goat polyclonal anti-eIF4A1 (sc-14211; 1:1000) and mouse anti-eIF4A2 (sc-137147; 1:200 for immunofluorescence) were from Santa Cruz Biotechnology. Rabbit anti-eIF4A2 (Ab194471; 1:3000) and rabbit anti-TIA-1 (Ab40693; 1:400) were from Abcam. Secondary antibodies used were: horseradish peroxidase (HRP)-conjugated goat anti-rabbit-IgG and rabbit anti-mouse-IgG (Dako), both used at 1:2500 for western blotting, FITC-conjugated anti-mouse-IgG and TritC-conjugated anti-rabbit-IgG (Sigma-Aldrich), Cy5-conjugated anti-rabbit-IgG (Life Technologies) and TritC-conjugated anti-goat-IgG (Santa Cruz Biotechnology) all used at 1:200.

Rat colons were embedded in Technovit 7100, and tissue sections of 7 μm thickness were cut with Reichert Jung 1140 Autocut microtome for immunofluorescent staining. Tissue sections were then blocked for 20 minutes in PBS (Gibco) containing 5% fetal bovine serum (FBS; Gibco) and 0.1% TritonX (Sigma). Next, sections were incubated overnight with mouse anti-Axl (Santa Cruz) primary antibody (1 : 100 dilution) or isotype-matched negative control antibody, washed three times with PBT (PBS containing TritonX) for 10 min each, incubated with FITC conjugated anti-mouse IgG (Sigma) secondary antibody (1 : 250 dilution) for 90 minutes, and washed three times with PBT (10 minutes each). Finally, samples were washed with PBS containing 4′,6-diamidino-2-phenylindole (DAPI; 1 : 10000 dilution) and mounted in Citifluor mounting media (Citifluor Ltd.). Samples were analyzed using epifluorescent illumination of the Axiovision Z1 fluorescent microscope (Zeiss), and images recorded by Axiovision software.

The VK2/E6E7 cells were grown on glass coverslips, fixed with 4% paraformaldehyde for 40 min, and then permeabilized with 0.1% Triton X-100 for 20 min at room temperature (RT). Cells were then blocked with 3% bovine serum albumin (Sigma-Aldrich) for 2 h to block non-specific antibody binding and then incubated overnight at 4 °C with antibody against HBD-1 (Abcam, Cambridge, UK). After washing, cells were incubated with FITC-conjugated anti-mouse IgG (1:1000 dilution; Sigma-Aldrich) for 1 h at RT, and DNA was counterstained with propidium iodide (Sigma-Aldrich). Coverslips were mounted with Fluorescence Mounting Medium (DAKO). Fluorescence was detected by confocal laser microscopy using a Zeiss LSM510 instrument (Zeiss, Jena, Germany).

IF antibody assays were performed as previously described [8 (link)]. Cultured cells were placed on slides, treated with fixing solution I (4% paraformaldehyde plus 400 mM sucrose in PBS) and held at 37°C for 30 min. Slides were then treated with fixing solution II (fixing solution I plus 0.5% Triton X-100) and held at room temperature for 15 min. After washing with PBS, slides were treated with a blocking buffer (0.5% BSA in PBS) at room temperature for 1 h and washed three additional times with PBS prior to reacting with various primary antibodies (anti-Nestin [abcam] or anti-Tuj1 [GeneTex]) at 1:100 dilutions, either overnight at 4°C or for 1 h at 37°C. Slides were washed five times with cold PBS before reacting with FITC-conjugated anti-mouse IgG or TRITCconjugated anti-rabbit IgG (Sigma-Aldrich). After four more washes with cold PBS, slides were mounted and observed using a fluorescence microscope. DNA was stained with DAPI (Sigma- Aldrich) to localize nuclei.

Antibody-binding assay was performed as previously described [32 (link)]. Briefly, pneumococci were plated on blood agar for overnight growth, then cultured in THY to OD 600 nm 0.4–0.5 (~10⁸ CFU/mL) and harvested by centrifugation. Bacteria were washed, suspended in PBS, and incubated with 1% of individual sera for 30 min at 37 °C. Samples were washed once with PBS before incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Sigma) for 30 min on ice. Samples were fixed with 2% formaldehyde after two washing steps and stored at 4 °C. Flow cytometry analysis was conducted using FACSCanto (BD Biosciences), and 10,000 gated events were recorded. Mean fluorescence intensity (MFI) was determined for each sample.