sGAG content of cricothyroid muscle and cricoid cartilage from three randomly selected larynges was quantified using Blyscan™ sGAG Assay (Biocolor, Northern Irland, UK). This assay is widely used to measure sGAG content in cartilages, organs/tissues (such as heart, lungs, kidney, larynx, and trachea), and solid tumors.12 (link)–14 (link) The assay was performed following the manufacturer’s protocol. In summary, the weight of each of the muscle and cartilage specimens (3 each) was recorded. The sGAG was extracted from the tissue using a Papain extraction reagent. Then, a dye was added that forms a complex with sGAG. The dye-bound sGAG was precipitated and drained. Subsequently, a dissociation agent was added to unbind the sGAG from the dye. The recovered sGAG content was measured at 656 nm using spectrophotometry and estimated by plotting on a standardized curve.