HUVEC were harvested after light trypsinization, washed in cold PBS, suspended in 1 ml cold blocking buffer (PBS/1% FBS) that contained 1 ug first antibody [8G4, mouse anti-human E-selectin antibody (IgG1 subtype) from Novus (Littleton, CO), or mouse IgG1], and incubated on ice for 1h with occasional agitation. Cells were then centrifuged (500xg, 5 min), washed 3x with cold PBS, incubated with 100 ng/ml second antibody (Alexa-488 labeled donkey anti-mouse IgG, Life Technology), and washed 3x with cold PBS. Cell suspensions were analyzed with FACScan (Becton Dickinson, San Jose CA). 104 events were collected for each analysis. All flow cytometry histograms were representative selections from at least three separate experiments
Alexa 488 labeled donkey anti mouse igg
Manufactured by Thermo Fisher Scientific
Alexa-488 labeled donkey anti-mouse IgG is a secondary antibody used in immunoassays and immunochemical procedures. It binds to mouse immunoglobulin G (IgG) and is conjugated with the fluorescent dye Alexa Fluor 488, which can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Facscan, by BD (2 mentions)
Alexa 568 labeled donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti cd11b, by Bio-Rad (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Fluoview10, by Olympus (1 mentions)
Anti neun, by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti vimentin, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa 488 labeled donkey anti mouse igg
1
Flow Cytometry Analysis of E-selectin
2
Cell Surface Antigen Binding Assay
3
Quantifying Kinase Activity in Glia Cells
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The sections were gently washed in PBST for 3 times and blocked with 5% fetal horse serum diluted in PBST for 1.5 h at room temperature, followed by treatment with a cocktail of 2 antibodies from anti-p-Erk1/2 (1:300, Cell Signaling), anti-p-p38 (1:300, Cell Signaling), anti-NeuN (1:2000, Millipore), anti-CD11b (1:1000, Serotec), and anti-glial fibrillary acidic protein (GFAP; 1:1000, BD Bioscience) antibodies for 20 h at room temperature. The sections were then treated with Alexa® 488-labeled donkey anti-mouse IgG and Alexa® 568-labeled donkey anti-rabbit IgG (1:200, Invitrogen) cocktails for 3 h at room temperature in the dark. Exposure to light was reduced to minimum after mounting and coverslip, antifade reagent (ProLong® Gold, Invitrogen) was used to maintain fluorescence until microscopy. To assess the pERK and pp38 expression level in the microglia and astrocyte, the intensity of pERK and pp38 were measured on a fluorescent microscopic picture of 400× magnifications. Region of interest (ROI) in the hippocampus were drawn along the CD11b-positive microglia and GFAP-positive astrocyte. Intensity of each ROI was measured by Fluoview 10 (Olympus, Japan).
4
Immunofluorescence Analysis of Cell Markers
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Identical samples to those used for histological examinations were employed. For antigen retrieval, sections were soaked in 10 mM citric acid (pH 6) at 95 C for 20 min. After permeabilization with 0.2% Triton-X in PBS and blocking with 1% bovine serum albumin in PBS, the sections were incubated overnight at 4 C with primary antibodies, 1:500 dilution of a mouse monoclonal anti-asmooth muscle actin (SMA) (Dako, M0851, Carpinteria, California) and 1:200 dilution of a rabbit monoclonal anti-vimentin (Cell Signaling, D21H3, Danvers, MA). The sections were then incubated for 1 h at 25 C with the following secondary antibodies: 1:500 dilution of Alexa 488-labeled donkey anti-mouse IgG (Invitrogen, Carlsbad, CA) and 1:500 dilution of Alexa 568-labeled donkey antirabbit IgG (Invitrogen). Cell nuclei were counterstained with 4',6diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen). Omission of the primary antibodies served as a negative control.
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