Two Separated Protocols with the Most Important Comments for Skeletal Staining in Embryonic and Adulthood Period in Laboratory Animals

Skeletal staining is an important part of anatomical studies which can evaluate macroscopic disorders in bones and cartilages development. The aim of the present article is to illustrate two distinct protocols by all details in order to stain skeletal system of the lab animals in all ages of their lives so it becomes easy for future studies in this field. In this paper, staining method was based on sample’s ages. So, all the steps of double skeletal staining described in 2 protocols, one for embryos and newborns and another for adults. Fixing by ethanol, alcian blue staining, dehydration by ethanol, alizarin red staining, washing by potassium hydroxide and glycerol, storage in pure glycerol were the stages of embryos and newborns skeletal staining. Fixing by neutral formalin buffer, placing the samples in ddH2O, fixing again by ethanol, skinning and eviscerating, alcian blue staining, trypsin digestion, alizarin red staining, potassium hydroxide clearing, KOH and glycerol clearing and finally storing the samples in pure glycerol were the steps of adults staining. Outcomes of a good and reliable procedure for skeletal staining with alizarin red and alcian blue are the samples whose cartilages and bones are stained blue and red respectively. They are completely transparent so that the skeletal parts can be seen through them. It is shown that skeletal staining procedures differ in embryos and adults although in the most surveys were tried to use a same protocol in diferent ages of laboratory animals.