Safranin O (red) stains proteoglycans, fast green (green) stains background, and hematoxylin stains nuclei in cartilages. The cartilage is stained orange to red, and the nuclei are stained black. In brief, deparaffinized samples were hydrated, then the slides were immersed in Weigert's hematoxylin for 5 min, followed by rinsing with distilled water for 2 min until excess stops leaching out of tissue. The sections were dipped once in 1% acid alcohol for 2 s followed by gently rinsing in distill water for 2 min. The sections were then further stained in 0.02% fast green solution for 30 s, and dipped in 1% acetic acid for 30 s and excess liquid was absorbed using paper towel. Subsequently, the sections were stained with Safranin O solution for 10 min followed by rinsing in 95% ethanol. After dehydrating with three changes of 95% ethanol, two changes of 100% ethanol, three changes of xylene (1 min each), added 1–3 drops of Cytoseal 60 mounting medium and a cover slip was placed over the stained tissue.
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