Comparison of cartilage and bone staining methods based on using acid and acid-free protocols for study of ontogeny in common Bream (Abramis brama)

The traditional method for staining cartilage and bone in vertebrates is to use Alcian blue and Alizarin red. Acid is used in the base of the ingredients of Alcian Blue solution, which led to clear distinguishing of the stained skeletal structures as much as possible. But the problem is that if acid is used, some of the bone ions are removed leading to errors in the results. Therefore, the present study aimed to compare two using acid and acid-free protocols for staining cartilage and bone during the early development of Common bream (Abramis brama) to provide a solution in future studies.

For staining, we applied acid-based (acetic acid) and acid-free protocols using Alican blue and Alizarin red during the processing stages of watering, staining of cartilage and bone, neutralization, watering, protein digestion, color removal, and washing and clearing.

Based on the results, it can be stated that in studies of the early development of bony fishes, the use of cartilage and bone staining method based on the acid-free protocol is more suitable than using acid because this method helps to better understand the sequence and pattern of skeletal ontogeny more accurately during the early development period.