4.9. Alcian Blue Staining and Quantification

Alcian Blue staining was performed by fixing micromass cultures at day 3 and then incubating them with 0.1% Alcian Blue, pH 2.5, for 2 h. Quantification of the staining was achieved after extensive washing with water by extraction with 6 M guanidine-hydrogen chloride for 8 h at room temperature. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dye concentrations were determined spectrophotometrically at A₆₃₀ [40].