4.4.2. FAST Staining

To study the glycosaminoglycan (GAG) contents in IVD, a multi-dye histological staining using Fast green, Alcian blue, Saffranin-O, and tartrazine was performed on IVD tissue sections accordingly [53]. In brief, parafilm embedded tissue sections were first dewaxed in xylene and then rehydrated in a stepwise gradient of ethanol. The IVD sections were first stained with 1% Alcian blue 8GX (A3157, Sigma) pH 1.0 for 1 min, followed by 0.1% Saffranin-O (S8884, Sigma) for 3 min. Saffranin-O reddish colour differentiation was performed in 25% ethanol for 15 s. The tissue sections were then stained in 0.08% Tartrazine (T0388, Sigma) with 0.25% acetic acid for 45 s and finally counterstained by 0.01% Fast green (F7258, Sigma) solution for 5 min. Sections were finally air-dried, mounted in DePeX (BDH Laboratory; Poole, UK), and examined under a Nikon Eclipse 80i microscope (Tokyo, Japan).
