Alcian blue binds to the proteoglycans components of the extracellular matrix in chondrocytes and turns the cartilage color to blue. Alizarin red stains free calcium and certain calcium compound a red or light purple color. This procedure is a useful tool to study the skeletal development in several fish species and then to precociously diagnose malformations in cartilage and bone development in small larvae and eventually to better characterize the effect of different environmental and/or nutritional factors on the ossification status of specific skeletal components. It should be noted that according to different species, the corrections in the protocol are still reported. In this experiment 520 specimens of rainbow trout in different life stages from one-day post hatching (dph) to 30 days, 1.5 ±0.1 and 3.5±0.5 cm in length were studied. The double staining protocol for developing rainbow trout was defined based on diverse published protocols. For an optimal staining protocol design, the incubation times of the different solutions were adjusted. Then the photos of stained specimens will be used to study and compare. The detection of cartilage and bone helps to understand the ontogeny of fish skeletal structure and the effects of environmental factors on bone density and plasticity. In addition, given the importance of skeletal and muscle\\\'s interactions, it is possible to conduct a survey on specific factors to change the skeletal tissue.

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