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gentle shaking. Blots were washed 3 times (5 min/wash) in PBS solution containing 0.05% Tween-20 and incubated for 1 hour with horseradish peroxidase–conjugated swine antibodies raised against rabbit IgG k (diluted 1:5,000 in blocking solution); blots

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in American Journal of Veterinary Research

measured with a specific sandwich ELISA 8 in which the primary antibody (rabbit anti-MPO IgG) was coated onto microplate c wells. For standard preparation, MPO was extracted from equine neutrophils isolated from whole blood by sedimentation on density

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in American Journal of Veterinary Research

-BrdU monoclonal antibody followed by incubation with a Cy3 dye-conjugated affinity-purified goat anti-mouse IgG e at a ratio of 1:1,000. The percentage of BrdU-positive nuclei was determined in comparison to the total cell population as marked by a nuclear stain

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in American Journal of Veterinary Research

as a negative control sample. After incubation, slides were rinsed and biotinylated anti-rabbit mouse IgG secondary antibodies j were applied and allowed to incubate for 10 minutes. Slides were rinsed, and an immunoperoxidase system k was applied to

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in American Journal of Veterinary Research

240/244 (1:2,000 dilutions). Rabbit monoclonal antibodies i specific to total 4EBP1 and 4EBP1 at Thr 37/46 (1:1,000 dilutions) and total rpS6 (1:10,000 dilution) were also used. The secondary antibody used was a goat anti-rabbit IgG (heavy and light

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in American Journal of Veterinary Research

:1,000 dilution) and total rpS6 (1:10,000 dilution) were also used as primary antibodies. A goat anti-rabbit IgG conjugated with horseradish peroxidase q (1:10,000 dilution) was used as the secondary antibody. All antibodies had been previously used to measure

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in American Journal of Veterinary Research

:1,000 dilution for each antibody), k and polyclonal rabbit anti-mouse antibodies that recognized total (1:2,000 dilution) k and phosphorylated Ser 473 (1:2,000 dilution) k Akt. Following washing, membranes were incubated with a goat anti-rabbit IgG (H+L) with

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in American Journal of Veterinary Research