Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

Figure 4

Histochemical stainings of cartilage, bone, and myelinated nerves of chicken embryos revealed normal chondrogenesis, osteogenesis, and myelination of the nerves. Chicken embryos cultivated either in ovo or ex ovo were sacrificed at E5, E10, E13, and E15 and subsequently fixed using 4% PFA. Afterwards, specimen was skinned and eviscerated followed by staining at 37°C overnight. Destaining in a graded series of ethanol was followed by clearing in 1 : 2 BABB/ethanol, 2 : 1 BABB/ethanol, and 100% BABB (1 : 2 benzyl alcohol/benzyl benzoate). Upper panel: 0.3% Alcian Blue staining solution in 70% ethanol and addition of 5% acetic acid for cartilage at E5, E10, E13, and E15 of in ovo- and ex ovo-cultivated chicken embryos. Middle panel: comparison of osteogenesis at E5, E10, E13, and E15 using 0.1% Alizarin Red S staining in 95% ethanol. Lower panel: staining of lipids using 5% saturated Sudan Black B in 70% ethanol indicates myelination of the optic nerves in E13 and E15. Arrowheads show the ending of optic nerves in the blowup.