Alexa fluor 647 goat anti mouse igg antibody

Zebrafish larvae were fixed at 4 °C overnight in 4% PFA in PBST (PBS, 0.1% Triton X-100). The samples were then washed with PBST and incubated in acetone at −20 °C. Larvae were washed once with PBST and PBSDT (PBS, 1% BSA, 1% DMSO, 1% Triton X-100), and incubated in 5% goat serum (Vector) in PBS-DT at room temperature (RT) for 2 h. The samples were incubated with the primary antibody solution (anti-zebrafish Parvalbumin7 antibody, 1/1000, a marker for Purkinje cells in zebrafish [38 (link)]) at 4 °C overnight. After six washes with PBST, the samples were incubated with the secondary antibody (Alexa Fluor 647 goat anti-mouse IgG antibody (Molecular Probes, 1/500)). Nuclei were counterstained with DAPI (Molecular Probes). These specimens were observed using a confocal microscope.

Leukocytes isolated from three individual fish were treated as three independent samples. The isolated leukocytes were suspended in serum-free L-15 medium, and transferred to 24-well plates (500 μl/well) at 5 × 10⁶ cells per well. The leukocytes were incubated with Japanese flounder rIFN I-3 (50 ng/well, at a final concentration of 0.1 μg/ml) for 12 h at 22°C. The pET-28a-derived his-tag protein was added to the negative control wells. Leukocytes were labeled with the anti-Japanese flounder IgM mAb and the secondary Alexa Fluor® 647 goat anti-mouse IgG antibody (Invitrogen/Molecular Probes, USA). Finally, intracellular reactive oxygen species (ROS) were detected using the ROS assay kit (Beyotime, China), according to the manufacturer's instructions with some modifications. Briefly, the cells were cultured in serum-free L-15 medium containing 0.1% 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 22°C for 30 min, prior to being washed with serum-free L-15 medium three times and measurement on the BD Accuri C6 flow cytometer. The fluorescence intensity of DCFH-DA was detected to assess intracellular mIgM⁺ B lymphocyte ROS levels.

Validation of the 2-DE data was carried out using western blot (WB) analysis. To assure the reproducibility of the WB analysis, at least three experimental replicates from each patient’s serum were performed. Proteins (40 μg) were separated by SDS-PAGE and transferred to Hybond® ECL nitrocellulose membrane (GE Healthcare, Freiburg, Germany). Immunodetection was performed as described previously [21 (link)]. Briefly, membranes were blocked in 5% milk for 2 hours at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibody including polyclonal rabbit anti-human haptoglobin (Hp; 1:1,000), rabbit anti-human apolipoprotein (Apo) C-III antibody (Genway, San Diego, CA, USA), rabbit anti-human ApoA-II antibody (Genway) or a rabbit anti-human vitamin D-binding protein antibody (Abcam, Cambridge, UK).
After washing three times in Tris-buffered saline–Tween buffer, nitrocellulose membranes were incubated with the corresponding secondary antibody (Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG, 1:2,000; Molecular Probes, Darmstadt, Germany). Air-dried blot membranes were then scanned at 50 μm resolution on a Fuji FLA-5100 scanner with single laser-emitting excitation light at 635 nm.

Proteins (150 μg) were separated by isoelectric focusing and SDS-PAGE as described above and transferred to Hybond® ECL nitrocellulose membrane. Immunodetection was performed as described above. Mouse monoclonal anti-vitamin D-binding protein (1:1,000; Abcam) was used as primary antibody. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody (1:2,000) was used as secondary antibody.

The validation of the 2-DE data was carried out using Western blot analysis. To assure for the reproducibility of the Western blot analysis, at least three biological and experimental replicates were performed. 40 μg proteins were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (GE Healthcare). Immunodetection was performed according to Towbin et al. [24 (link)]. Briefly, membranes were blocked in 5% milk for 2 h at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibody. Mouse monoclonal anti-CRABP1 (1:1000) (abcam), rabbit anti-Cofilin (1:1000) (sigma), mouse monoclonal anti-StAR (1:250) (abcam) and mouse monoclonal anti-ß-actin (1:5000) (sigma) were used as primary antibodies. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG (1:2000) were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 μm resolution on a Fuji FLA-5100 scanner (Fuji Photo) with single laser-emitting excitation light at 635 nm.

Proteins (40 μg) were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (GE Healthcare). Immunodetection was performed according to Towbin et al.71 (link). Mouse monoclonal anti-Trim28/Kap1 [20C1] antibody ChIP Grade (Abcam), rabbit monoclonal anti- Cbx5/HP1 alpha antibody (Abcam)/ goat polyclonal to Cbx5/HP1 alpha-ChIP Grade (Abcam), rabbit polyclonal to Cbx1/HP1 beta (Abcam), rabbit polyclonal to Cbx3/HP1 gamma (Abcam), mouse monoclonal anti-ß-actin (Sigma) (1:5000), rabbit polyclonal anti-H3K9me3 ChIP grade (Abcam) and Gapdh (HyTest, Turku, Finland) were used as primary antibodies. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG (1:2000) were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 μm resolution on a Fuji FLA-5100 scanner (Fuji Photo) with single laser-emitting excitation light at 635 nm.

Western blot analysis was carried out to confirm the proteomics data and to investigate the effect of the cytokine/hormone treatments on the level of the protein of interest. For our Western blot analysis the protocol of Towbin et al. [26 (link),27 (link)] was adapted. First, 50 µg of the cell extracts or secretome proteins were subjected to a denaturation step with Laemmli buffer and loaded on a 12% SDS-gel. Rabbit anti-fibronectin (FN1) (Sigma), rabbit anti-alpha smooth muscle actin (Abcam), mouse anti-peptidyl-prolyl cis-trans isomerase A (PPIA) (Abcam), and mouse anti-β-actin (Sigma), as the primary antibodies were diluted in blocking buffer, then added to the membrane and allowed to incubate overnight at 4 °C. Molecular Probes^® Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 µm resolution on a Fuji FLA-5100 scanner (Fuji Photo, Kanagawa, Japan) with single laser emitting excitation light.