Medicool

Bovine embryos were incubated with nfeAFP11 labelled with the fluorescent probe Alexa 488 (Invitrogen, Carlsbad, CA, USA) in phosphate buffered saline (PBS) at 4–5 C (Medicool, Sanyo, Osaka, Japan), 19–23 C (room temperature, RT) or 37 C (MIR–162 incubator, Sanyo) for 10, 20, 30 and 60 min. After incubation, the embryos were washed several times in PBS supplemented with 5% FBS (MP Biomedicals, Illkirch, France) and mounted under cover slips without compression in medium containing 50% glycerol and 25 mg/ml of sodium azide. Interaction between bovine embryos and nfeAFP11 was observed using a Leica DM LD fluorescence microscope (Leica, Wetzlar, Germany) at an excitation/emission wavelength of 485/530 nm. The morphology of the chilled embryos was evaluated using light microscopy (Leica Wild M3Z, Leica), and all the photomicroscope images were recorded using an Olympus DP21 microscope digital camera (Olympus, Tokyo, Japan). Individual mean pixel intensity was measured on a
per embryo basis using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Furthermore, a Leica DM IRE2 confocal microscope system (Leica) was also used to observe nfeAFP11-embryo interactions. The embryos were incubated with nfeAFP11 labelled with Alexa 488 in PBS at RT or 37 C for 30 min. All the confocal microscope images were recorded using a 3CCD color video camera (Sony, Tokyo, Japan).