Efficient Detection of Indian Hedgehog During Endochondral Ossification by Whole-Mount Immunofluorescence

Endochondral ossification is a process essential for the formation of the vertebrate skeleton. Indian Hedgehog (IHH) is a key regulator of this process. So far, monitoring IHH expression in whole-mount developing skeletal structures has been hampered by the permeability and the opacity of the tissue. Whole-mount preparations require advanced techniques of fixation, clearing, and staining. We describe a reliable method for fixing, immunostaining, and clearing whole-mount developing cartilages that allows for the detection of IHH in the developing skeleton of avian embryos. The fixation process ensures a proper preservation of cellular structures and, especially, the antigenicity of the tissue, allowing the antibody labelling of IHH. This protocol reveals specific cell staining in localized regions of the developing cartilage, facilitating the study of IHH function during key periods of skeletogenesis.