Flk-1lacZ conceptuses were fixed in 4% paraformaldehyde (2 hr, 4°C) and stained for X-gal activity as previously described (Downs and Harmann,1997), but for 6 hr only, after which specimens were rinsed in phosphate-buffered saline (PBS, Sigma) and dehydrated in increasing methanols as described below prior to immunostaining.
Immunostaining Bouin's- and 4% paraformaldehyde-fixed and sectioned specimens was as previously described (Inman and Downs,2006a) using primary antibody to Oct-3/4 (N-19, goat polyclonal SC-8628, Santa Cruz Biotechnologies, Santa Cruz, CA) at dilutions of 1/67, 1/100, and 1/250 for 1.5 and 3.0 hr at room temperature, and 18 hr at 4°C. For whole mount immunohistochemistry, all conceptuses were fixed in 4% paraformaldehyde (2 hr, 4°C), and rinsed in phosphate-buffered saline (PBS, Sigma), followed by dehydration in an increasing series of methanols, and indefinite storage at −20°C in absolute methanol. Just prior to immunostaining, the yolk sac was opened up in all specimens ≥ OB stage by gentle tearing with a forceps. For immunostaining, all steps were carried out on a rocking platform at room temperature, with the exception that primary and secondary antibody binding were carried out at 4°C on the rocker. Endogenous hydrogen peroxidase activity was eliminated with 5% hydrogen peroxidase/methanol for 5 hr. This was followed by blocking donkey non-specific antigen sites for 2 hr in PBS containing 5% donkey serum (Chemicon) and 0.1% Triton-X (“PBSST”). Specimens were incubated overnight at 4°C in primary antibody against Oct-3/4 (1/100 dilution in PBSST), and the next day, they were washed 5 times in PBSST for a total period of 5 hr, followed by application of secondary antibody (1/500 dilution in PBSST; biotinylated donkey anti-goat IgG, SC-2042; Santa Cruz Biotechnologies, Santa Cruz, CA) and incubation overnight at 4°C. After 5 hr of rinsing in PBSST, specimens were incubated for 3 hr in ready-to-use ABC reagent (PK-7100, Vector Labs, Burlingame, CA), washed 3 times during 1.5 hr in PBSST, then twice during 20 min in PBT, in which bovine serum albumen (0.02%, Sigma, A-4378) replaced the donkey serum. The antibody complex was visualized with diaminobenzoate chromagen (DAKO Corporation, Carpinteria, CA) for 5 min at room temperature, after which specimens were fixed again in paraformaldehyde at 4°C overnight, oriented transversely, sagitally, or frontally, and embedded, after which they were sectioned at a thickness of 6 μm, dewaxed, counterstained in hematoxylin, coverslipped, and examined in the compound microscope. Control experiments included elimination of antibody, and pre-binding the Oct-3/4 antibody with Oct-3/4 peptide (SC-2042p, Santa Cruz Biotechnologies) for 10 hr at 4°C.