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a high CD4+:CD8+ ratio (T-helper:T-cytotoxic ratio), 4–7 whereas nondiseased pericorneal tissue (conjunctiva and lacrimal gland) is populated predominantly by T-cytotoxic cells. 8 In addition to T cells, antibodies (IgG, IgM, or IgA) cause local

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in American Journal of Veterinary Research

secondary antibody (goat anti-dog IgG e diluted 1:10,000 in blocking buffer) for 15 minutes. Bands were made visible by reaction with enhanced chemiluminescence reagents followed by exposure to x-ray film. As a negative control sample, 1 strip/blot was

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in American Journal of Veterinary Research

(diluted 1:250 to 1:500). Sections then were washed and incubated in the appropriate secondary antibody. Secondary antibody for GFAP and HIF-1α was biotinylated goat anti-rabbit IgG, e whereas the secondary antibody for HSP 60 was biotinylated horse anti

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in American Journal of Veterinary Research

at 22°C. The primary antibody was a mouse monoclonal antibody directed against the rat COX-2 protein. c A purified mouse IgG 1 isotype d control was used for negative controls. Both antibodies were used at a dilution of 1:30 in PBS solution

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in American Journal of Veterinary Research

, which may have caused nonspecific antibody binding on the western blot. However, this would be expected to cause reaction at the light-chain (25 kd) and heavy-chain (50 kd) sites for IgG and to cause similar bands on the negative control attributable to

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in American Journal of Veterinary Research

incubated with mouse anti-BrdU monoclonal antibody (1:200 dilution) for 1 hour. This was followed by another series of washes and incubation with peroxidase goat anti-mouse IgG antibody (1:2,000 dilution) for 30 minutes. After another series of washes

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in American Journal of Veterinary Research

intraocular antibody production. However, other studies 15,24,32,34,35 suggest a more stringent C-value of > 3 or 4 to definitively demonstrate intraocular antibody production. Immunoblotting for local specific IgG and IgA may have increased the sensitivity

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in American Journal of Veterinary Research