Modified double skeletal staining protocols with Alizarin red and Alcian blue in laboratory animals

Skeletal staining is a way to study the effect of many chemical or herbal substances on development of bones and cartilages in order to record the level of probable deformity.This article aimed to present a modified protocol to make all skeletal studies on laboratory animals possible. The important notes about skeletal staining were fixing by ethanol and clearing by potassium hydroxide in embryonic and newborn samples. In adults it was fixing by neutral formalin buffer, then ethanol after washing the samples in ddH2O, and clearing the samples by trypsin and potassium hydroxide in separated stages. The amount of colors used for cartilages and bones was differentin animals with different ages for a good stained sample. The mentioned procedures resulted in completely clear stained samples whose skeletal parts, i.e. cartilages and bones,were stained blue and red, respectively. Although most reviewed investigations have used the same protocol in different laboratory animals with different ages, the used materials and also their concentrations for skeletal staining procedures differ in embryos and adults.