Figure 2

Oct-4 expression in an in vitro-derived d10 bovine embryo. Strong Oct-4 nuclear immunofluorescence (left panel; red) is detected in the i.c.m. at the left pole of the embryo, while weaker, more diffuse immunofluorescence is present over trophectoderm. DAPI nuclear staining (blue) is shown in the center panel. In the right panel, Oct-4 (red) and DAPI (blue) signals have been merged. The merged image indicates that, whereas all nuclei of the i.c.m. are strongly Oct-4 positive, Oct-4 signals are weaker and more variable over trophectoderm, with some cells apparently Oct-4 negative. Controls performed with a non-relevant IgG failed to show nuclear staining (not shown). Positive controls (also not shown) with the anti-Oct-4 IgG provided nuclear staining in undifferentiated F9 embryonic carcinoma cells but not in JAr choriocarcinoma cells (data not shown). The bovine embryos were fixed with 2% paraformaldehyde-PBS for 30 min at room temperature, permeabilized with 1% Triton X-100 for 30 min, and incubated overnight at 4 C with primary antibody (affinity purified rabbit anti-Oct-4 IgG in PBS; T.E., R.M.R. unpublished) at a concentration of 4 ng/μl. After washing, the blastocysts were exposed to secondary antibody (goat anti-rabbit IgG conjugated with Alexa Fluor 568; Molecular Probes, Eugene, OR) diluted 1:1000. Nuclear staining was performed with DAPI (Sigma, St. Louis, MO) at a concentration of 5 ng/μl. Bars represent 100 μm. Images were captured by using a Nikon Eclipse 800 microscope equipped with a CoolSnap HQ RTE/CCD 1217 digital camera operated by MetaMorph 4.6 software (Universal Imaging Corp., Downington, PA) and edited by Adobe Photoshop 6.0.