Antibodies and Recombinant Ovine IFN-т
A monoclonal mouse antibody against recombinant bovine GM-CSF, GM-CSF17.2, was purchased from VMRD (Pullman, WA). The negative control, mouse immunoglobulins G1 (IgG1), was acquired from Dako Diagnostics (Mississauga, ON, Canada), and the biotinylated goat-anti-mouse secondary antibody was obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Polyclonal rabbit antisera against ovine COX-1 (COX-1 241) and COX-2 (COX-2 243) were donated by Merck Frosst (Kirkland, QC, Canada). Biotinylated goat-anti-rabbit secondary antibody was obtained from Dako Diagnostics. Recombinant ovine IFN-т was kindly donated by Dr. Fuller W. Bazer (College Station, TX). Its amino acid sequence is 80% homologous to bovine IFN-т and it cross-reacts in cattle.
In experiment 1, cryostat cross-sections (6 |xm) were prepared from frozen uteri with a Shandon SME cryostat (Fisher Scientific, Nepean, ON, Canada). IHC was performed twice on sections from a total of 30 blocks, each from 18 pregnant or 12 cyclic cows, with three animals per day postestrus. Two or three sections, each from different animals, were mounted on each SuperfrostPlus slide (Fisher Scientific) coated with 3-aminopropyltriethoxysilane (Sigma-Aldrich, Oakville, ON, Canada).