The procedure used for immunofluorescence microscopy of rat ovarian sections and porcine oocytes, zygotes, and blastocysts has been described in detail previously. Mouse monoclonal PCNA, polyclonal rabbit antiprohibitin, and polyclonal anti-rat cytochrome P450scc antibodies were used at a dilution of 1:200. The specificity of the antibodies was verified by incubating ovarian sections and oocytes, zygotes, and blastocysts without primary antibodies as well as with NRS.
After thorough rinsing, sections were mounted in glycerol containing 50 |xg/ml of л-pro-pyl gallate and then examined using either an Olympus BX41 microscope equipped with an Optronics MagnaFire digital camera and Prior Proscan motorized driven stage (Olympus, Melville, NY) or a Nikon Eclipse 800 microscope and CoolSnap HQ RTE/CCD 1217 digital camera (Roper Scientific, Tucson, AZ).
For digital image capturing, the exposure time was adjusted using sections incubated without the primary antibody to minimize any auto or nonspecific fluorescence recording without compromising the actual signal.