Bone and cartilage staining protocol for large avian specimens: Late‐stage embryonic emu ( Dromaius novaehollandiae ) and adult chickens ( Gallus gallus )

The ability to consistently and clearly visualize discrete anatomical parts of a specimen is crucial for biological research. For skeletal tissues, a classic and widely adopted technique uses a combination of Alizarin red for bone and Alcian or toluidine blue stains for cartilage. Published protocols for these stains have generally been optimized for small or prenatal samples. Adoption of these existing protocols on relatively large specimens, including somatically mature individuals and embryos of large-bodied taxa, has resulted in non-specific staining and insufficient clearing of extraneous tissue. This failure may be partly due to larger and denser tissues that reduce the penetration potential and speed of reagents. Here, we present a refined, detailed clearing and staining protocol, accompanied with photographs, optimized for larger avian specimens. Adult domestic chickens (Gallus gallus domesticus) and late-stage, embryonic emus (Dromaius novaehollandiae) were used to develop and test the effectiveness of the procedure. The latter exhibit larger and relatively mature soft tissue compared to those of late-stage embryos of smaller vertebrate taxa such as domestic chickens and mice. We found that the combined Alcian blue and Alizarin red staining works best on freshly frozen specimens compared to those fixed in formalin, and effective clearing and staining were possible without the use of tissue digestion reagents, such as trypsin and pancreatin. This step-by-step guide provides researchers with a consistent and effective skeletal staining protocol for larger vertebrates, allowing investigation of skeletal tissues at broader developmental and taxonomic scales, as well as for educational purposes.