Figure 5.

Nuclear and cytoplasmic staining of SOX2 protein in preimplantation embryos. (A–L) SOX2 antibody staining in wild-type blastocysts. Embryos were incubated in SOX2 (B,H,L), preimmune (PI; E), or OCT4 (K) antiserum. Nuclei were stained with 7-AAD (A,D,G) or DAPI (J). (C,F,I) Overlays of the antibody and 7-AAD staining to show colocalization in yellow. (A–F) Whole blastocysts. (G–I) An ICM isolated by immunosurgery. (J–L) Confocal images, using an affinity-purified SOX2 antibody. SOX2 is observed throughout the ICM in the nucleus (n) and cytoplasm (line) unlike OCT4, which is detected only in the nucleus. The arrowhead indicates the layer of ExEn where OCT4 is present but SOX2 levels are low. (M–V) SOX2 antibody staining in wild-type embryos from 1-cell to late morula (L-morula) stages. Embryos were stained for SOX2 using affinity-purified antibody (M–Q), and nuclei were visualized with DAPI (R–V). All panels are confocal images. n, nuclear staining; c, cytoplasmic staining; pb, polar body. (W) SOX2 antibody staining in blastocysts from Sox2^βgeo heterozygous intercrosses. Three separate litters were analyzed (1–3) using horseradish peroxidase and DAB staining. SOX2 is observed in all embryos. The last two embryos in each set are wild-type preimmune controls (*). Bars: A–F,J–L, 25 μm; G–I, 30 μm; M–V, 25 μm; W, 80 μm.