The procedure can be adjusted depending on the size/age of the specimens.ġKOH is highly caustic to skin. Concurrent with Alizarin red staining, exposure to potassium hydroxide (KOH) hydrolyzes soft tissue, leading to transparency and allowing visualization of stained skeletal elements. For further removal of fatty tissue and tissue permeabilization, specimens are exposed to acetone and then consecutively transferred to Alcian blue and Alizarin red staining solutions. They are then dehydrated and fixed in 95 % ethanol. Most laboratories use very similar protocols.Īs a brief overview of the process, specimens are prepared by removing skin, organs, and brown fat. The protocol described here is used in our laboratory. The protocol has been refined over the years, and there are variations employing enzymatic steps and different fixatives. The selective staining properties of Alcian blue and Alizarin red and their use in whole-mount skeletal preparation have been widespread for over a century. In contrast, because 99 % of the calcium in the body is localized in bone, Alizarin red is highly specific for bone. However, other tissues, such as the skin, contain GAGs and other glycoproteins that bind Alcian blue. Because cartilage contains higher concentrations of GAGs than any other tissue, it binds more Alcian blue. As a cationic dye, Alcian blue binds strongly to sulfated GAGs and glycoproteins, while Alizarin red, an anionic dye, binds to cationic metals such as calcium. Alcian blue and Alizarin red stain cartilage and bone, respectively. In contrast, during intramembranous ossification, mesenchymal cells differentiate directly into bone-forming osteoblasts. Chondrocytes undergo a process of stratified differentiation and apoptosis, enabling the replacement of cartilage by bone. These cells secrete a cartilaginous extracellular matrix rich in proteoglycans, glycosaminoglycans (GAGs), and collagen (types II and X). Endochondral bone formation occurs after cells in mesenchymal condensations differentiate into chondrocytes. The formation of bone occurs through two processes: endochondral and intramembranous.
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