polyclonal sheep anti cd62p p selectin primary antibody  (R&D Systems)

Journal: Nature communications

Article Title: Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63.

doi: 10.1038/ncomms4738

Figure Lengend Snippet: Figure 2 | Depletion of anxA8 reduces cell surface presentation of P-selectin. (a) HUVEC transfected as indicated were stimulated with histamine, incubated with fluorescently labelled anti-P-selectin antibodies and subsequently analysed by flow cytometry. MFIs of cell surface P-selectin of 10,000 cells were quantified. Statistical significance was evaluated by repeated measures ANOVA followed by Tukey’s multiple comparison test. **Po0.01; NS, not significant; bars represent means±s.e.m. from nine independent experiments. (b) Confocal imaging of histamine-induced cell surface P-selectin presentation of cells transfected as indicated (upper panel) or subjected to a second round of transfection using anxA8-specific siRNA together with a GFP expression vector (GFP) or a siRNA-insensitive anxA8 expression vector (anxA8i-GFP) 48 h after the initial transfection (lower panel). The cell surface pool of P-selectin was quantified by measuring MIFs in arbitrary units (AU). *Po0.05, ***Po0.001, P40.05 ¼ NS; one-way ANOVA with Tukey’s multiple comparison test. Data represent means±s.e.m. from at least 15 single cells from four independent experiments. Scale bars, 10 mm. (c) HUVEC transfected as indicated were stimulated with histamine. Untreated cells (non-transfected (NT)—w/o Ab) and cells treated with anti-P-selectin antibodies were analysed by AFM to visualize the surface topology. Representative 10 10 mm deflection images of fixed cells obtained in contact mode in fluid are shown. Scale bars, 1 mm. Note that antibody-decorated HUVEC transiently transfected with anxA8 siRNA (siA8) show a smoother and less granular surface compared with non-transfected HUVEC (NT), as well as cells transfected with non-targeting siRNA (siCt).

Article Snippet: Sheep polyclonal antibodies against P-selectin (AF 137; 1:300, 1:100 for surface staining, 25mg ml 1 for leukocyte adhesion assay), and sheep IgG isotype control (5-001-A) from R&D Systems were either used uncoupled or conjugated to FITC (Thermo Scientific; 8 ngml 1) or DyLight 488 (Thermo Scientific; 2.5 ngml 1).

Techniques: Transfection, Incubation, Cytometry, Comparison, Imaging, Expressing, Plasmid Preparation

Journal: Nature communications

Article Title: Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63.

doi: 10.1038/ncomms4738

Figure Lengend Snippet: Figure 3 | AnxA8 depletion leads to increased internalization of P-selectin from the cell surface. (a) HUVEC transfected with control (upper panel) or anxA8-specific siRNA (lower panel) were co-stained with antibodies recognizing P-selectin (red) and VWF (green). Draq5 was used to label the nuclei. Colocalization coefficients (of VWF with P-selectin) from at least 23 images from five independent experiments were determined. Data are means±s.e.m. Scale bars, 10 mm. *Po0.05; unpaired Student’s t-test. (b) Non-transfected HUVEC (NT) and HUVEC transfected with either non-targeting (siCt) or anxA8-specific siRNA (siA8) were cultivated for 48 h and then incubated in media containing antibodies against P-selectin for 24 h, fixed, permeabilized, incubated with the respective secondary antibodies and co-stained with antibodies against VWF. Scale bars, 10 mm. Colocalization coefficients of VWF with P-selectin antibodies from at least 13 individual cells from three independent experiments were determined as described in Methods. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. P40.05 ¼ NS. (c) HUVEC transiently transfected with either non- targeting siRNA or anxA8-specific siRNA were allowed to internalize FITC-coupled anti-P-selectin antibodies at 37 C for 30 min and subsequently analysed by flow cytometry. Relative fluorescence of internalized P-selectin was quantified and statistically analysed by unpaired Student’s t-test. *Po0.05; data are means±s.e.m. from four independent experiments (10,000 cells per experiment).

Article Snippet: Sheep polyclonal antibodies against P-selectin (AF 137; 1:300, 1:100 for surface staining, 25mg ml 1 for leukocyte adhesion assay), and sheep IgG isotype control (5-001-A) from R&D Systems were either used uncoupled or conjugated to FITC (Thermo Scientific; 8 ngml 1) or DyLight 488 (Thermo Scientific; 2.5 ngml 1).

Techniques: Transfection, Control, Staining, Incubation, Comparison, Cytometry