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with a sodium citrate antigen retrieval agent. b Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide, and nonspecific binding was blocked by subsequent incubation with a serum-free protein block. c Rabbit polyclonal IgG anti

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in American Journal of Veterinary Research

. Synovial fluid samples were incubated with hyaluronidase a (2,000 U/mg) at 37°C for 60 minutes to decrease viscosity. Twenty-five microliters of each serum and synovial fluid sample was albumin depleted with an immunoaffinity albumin and IgG depletion kit

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in American Journal of Veterinary Research

100 μL of an appropriate dilution of affinity-isolated rabbit anti-dog IgG (whole molecule) conjugated to horseradish peroxidase. j The conjugate was incubated for 1 hour at 37°C. Wells were again washed 4 times with washing solution, and binding of

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in American Journal of Veterinary Research

again before incubation with goat anti-rabbit IgG horseradish peroxidase conjugate antibody o (diluted 1:150) or goat anti-mouse IgG horseradish peroxidase conjugate antibody p (diluted 1:150) for 2 hours at room temperature. Primary antibodies were

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in American Journal of Veterinary Research

–conjugated goat anti-mouse IgG antibody diluted 1:10,000 in 2% skim milk in Tris-buffered saline solution containing 0.05% Tween 20 and developed by use of 5-bromo-4-chloro-3-indolyl phosphate nitroblue tetrazolium as a substrate. Serum COMP analysis —An

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in American Journal of Veterinary Research

-buffered saline (0.9% NaCl) solution containing 0.05% Tween and 1.5 g of nonfat dry milk. Then 30 μL of primary antibody (mouse anti-rat proteoglycan hyaluronan IgG monoclonal) was added, and the blot was incubated at 4°C overnight. The antibody was obtained from

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in American Journal of Veterinary Research

-collagen type I antibodies h (1:100 concentration) for 30 minutes; negative control slides were treated with goat IgG (1:1,000 concentration) instead of the primary antibody. Secondary and tertiary reagents in a labeled streptavidin biotin reagent system i were

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in American Journal of Veterinary Research

II collagen, C2C (COL2-3/4C long antibody) was assayed by use of an ELISA k to detect a neoepitope created by the specific cleavage of type II collagen by collagenases when degradation occurs. The assay used a mouse IgG antibody. CS 846 assay —To

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in American Journal of Veterinary Research

the plate was detected by use of goat anti-mouse IgG conjugated to alkaline phosphatase and addition of an alkaline phosphatase chromophore. After 1 hour, chromophore production was stopped by addition of 2M H 2 CO 3 (30 μL/well) and absorbance was

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in American Journal of Veterinary Research

.1% Triton X-100. r Cells were incubated with donkey anti-rabbit IgG labeled with green fluorescent dye s (diluted 1:100) for 2 hours at 23°C and then were washed 4 times with PBS solution. Cells were viewed on an inverted fluorescence microscope k

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in American Journal of Veterinary Research