The MCP-1 cDNA amplified by the PCR reaction described in the previous section was cloned into pGEM-T (Promega) using the manufacturer’s procedure. The insert was sequenced to determine its identity and orientation within the plasmid. The construct was linearized with NcoI and used as a template to generate 35S-labeled antisense RNA using SP6 DNA-dependent RNA polymerase. A sense RNA probe was prepared using T7 DNA-dependent RNA polymerase after linearization with NsiI. buy cheap antibiotics
In Situ Hybridization
In situ hybridization was performed on luteal tissue from a limited number of animals. CL were selected from the group of cows that had not undergone luteolysis (progesterone > 1 ng/ml at slaughter, n = 3) and from the group of cows that had undergone luteolysis (progesterone < 1 ng/ml at slaughter, n = 3). Four serial sections were cut (14 ^m) from each CL. Sections for in situ hybridization were dehydrated, fixed, and probed with 35S-labeled MCP-1 riboprobes. After the final high-stringency wash, the sections were dipped in autoradiographic K2 photographic emulsion (Ilford Ltd., Mobberley, Cheshire, UK) and exposed for 2 wk at 4°C. Sections were then developed (Kodak D-19; Eastman Kodak, Rochester, NY) and fixed (Ilford Hypam fixer; Ilford Ltd.) before staining in hematoxylin and eosin. The sections were finally mounted in DPX mountant before microscopic examination using both light-and darkfield illumination on a Nikon Microphot-SA microscope (Nikon Instruments, Garden City, NY).