The intensity of the in situ hybridization signal was analyzed using an NIH-Image analysis system (NIH, Bethes-da, MD). The number of graphic pixels occupied by silver grains (identified by a set gray threshold) within a defined area of the tissue section was counted and presented as a percentage of the total pixel number within the defined area. The hybridization intensity is therefore presented as the percentage of occupied pixels within a defined area of the tissue. Three serial sections were analyzed for each CL. Two were probed with the antisense RNA probe, and the remaining section was probed with the sense probe. Background hybridization intensity, measured with the sense RNA probes, was subtracted from the measurements obtained with the antisense probes to give the final hybridization signal. Within each CL, three separate fields were analyzed for each probe.
Immunohistochemistry was performed on sections of frozen luteal tissue serial to those used for in situ hybridization. T lymphocytes were identified in sections of luteal tissue using an avidin-biotin complex (ABC) method, modified as described by Cobb and Watson. The monoclonal antibody used was a specific marker for CD5 + T lymphocytes, CC17 (Institute for Animal Health, Compton, UK). This monoclonal antibody identifies mature T lymphocytes including CD8+ cytotoxic/suppressor T lymphocytes and CD4+ helper/inducer T lymphocytes. Immunohistochemistry was performed using a Vectastain ABC kit (Vector Laboratories, Peterborough, UK). The peroxidase substrate used to visualize the product was 3-ami-no-9-ethyl carbazole (Vector Laboratories). Sections of bovine lymph node were processed with each batch of CL as positive control samples, and negative controls were included in which the monoclonal antibody was replaced with normal mouse serum. antibiotic levaquin
MCP-1 mRNA expression, measured by in situ hybridization, was analyzed by ANOVA using a mixed model. The data were fitted to the model Y = ^ + Xi + Aj(i) + Bk(ij) + Cl(ijk) + ijklm, where Xi (fixed effect) represents CL collected before (i = 1) and after (i = 2) the onset of natural luteolysis, Aj represents cow number within treatments (random effect), Bk represents slide number within cows (random effect), and Cl represents replicate within slides (random effect). A similar procedure was performed for the reverse transcriptase data. In this case Bk represents the experiment number and Cl represents the replicate within experiments.