Histological Analysis of Cartilage Proteoglycans

Histological sections or intact μ-gels were stained with Alcian blue pH 1.0 (Rowley Biochemical) to identify sulfated proteoglycans22 (link). Immunofluorescence staining was performed following the cell membrane, HPG and nuclear staining described above. μ-gels were stained for aggrecan (1°: Abcam ab3778, 1:50 in PBS overnight at 4 °C; 2°: ThermoFisher AlexaFluor 546 goat anti-mouse, 1:200 in 5% BSA for 1 hr at room temperature). Additional μ-gels were stained for collagen II (1°: DSHB ii-ii6b3, 10 μg/mL in PBS overnight at 4 °C; 2°: ThermoFisher AlexaFluor 546 goat anti-mouse, 1:200 in 5% BSA for 1 hr at room temperature). Collagen II staining was performed with and without hyaluronidase digestion (300 μg/ml, 2.5 hours at room temperature).