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-human P-gp antibody m /million cells or 0.5 μg of antibody isotype control/million cells. Finally, incubation at 4°C in the dark for 20 minutes with fluorescein isothyocyanate conjugated rat anti-mouse IgG1 or IgG2a monoclonal antibodies n was performed

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in American Journal of Veterinary Research

antibody A-3 n was used to identify VEGFR-2 expression. Binding of primary antibodies was detected by use of fluorochrome-labeled secondary antibodies. o Polyclonal rabbit IgG p and monoclonal mouse IgG1 q served as isotype controls. Irradiated cells

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in American Journal of Veterinary Research

% BSA and 0.05% azide were labeled with the polyclonal rabbit anti-human tissue factor antibody d or rabbit IgG e (both at 20 μg/mL) for 15 minutes on ice, followed by a secondary Alexa488-conjugated goat anti-rabbit IgG c (1:200) for 15 minutes on

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in American Journal of Veterinary Research

/reaction) were resuspended in PBS with 1% bovine serum albumin d and 0.05% sodium azide (ie, reaction buffer) and were incubated with monoclonal mouse anti-dog TF antibody IgG (clone 133–2) 31 or mouse IgG isotype control q (each used at a final concentration

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in American Journal of Veterinary Research

primary antibodies against the polyprotein peptide tag, i p21, j MDM2, k or β-actin l for 1 hour; membranes then were incubated with secondary antibodies (HRP-labeled goat anti-mouse IgG m and HRP-labeled goat anti-rabbit IgG n ) for 1 hour ( Appendix

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in American Journal of Veterinary Research

with antibodies specific for caspase 3 q or β-actin r (both at a dilution of 1:500 in TBST and 5% bovine serum albumin) by incubation overnight at 4°C. Membranes were then washed and incubated with goat anti-rabbit IgG conjugated to horseradish

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in American Journal of Veterinary Research

-matched control antibodies (far-red fluorescent dye c -conjugated rat IgG2b [LO-DNP-11], d fluorescein isothiocyanate-conjugated mouse IgG1 [DAK-GO1], e and R-phycoerythrin-conjugated mouse IgG1 [W3/25] d ) were used for negative control staining. Following

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in American Journal of Veterinary Research

; and incubated overnight at 4°C. Membranes were then washed 3 times in TBST for 15 minutes, treated with goat anti-rabbit IgG conjugated to horseradish peroxidase aa at 1:15,000 in 2.5% milk in TBST for 1 hour, and washed 3 times. Antigen and antibody

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in American Journal of Veterinary Research

-month period. Flow cytometry The CMT25 or HMPOS cells (5 × 10 5 cells/reaction) were suspended in PBS solution with 1% bovine serum albumin and 0.05% sodium azide. Cells were labeled by incubation with mouse antiphosphatidylserine IgG1 o or

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in American Journal of Veterinary Research

, cells were incubated with fluorescein isothiocyanate–labeled anti-rabbit IgG gg (1:200) at room temperature for 1 hour; cells were counterstained with the double-stranded DNA-binding fluorescent dye as described. Photographic images of western blots

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in American Journal of Veterinary Research