Toluidine blue solution

All groups of TSs were cultured for 3 weeks, de-crosslinked, fixed with 4% paraformaldehyde, and embedded in paraffin using an automatic tissue processor (TP 1020, Leica, Germany). Next, TSs were gradually dehydrated in alcohol, cut into 8 μm sections, and placed onto charged slides. Sections were then dewaxed using Leica Autostainer XL (Leica, Germany) and underwent Toluidine Blue O and Picrosirius Red/Fast Green staining according to standard protocols for detection of GAGs and collagen, separately. Briefly, for Toluidine Blue O staining, sections were incubated in a Toluidine Blue solution (0.1% in DI water, Sigma Aldrich, MO) at room temperature for 2 min.^{[52 ]} The dye was then removed and samples were washed twice with DI water. After dehydration with ascending alcohol and clearing with xylene, coverslips were mounted to the slides using Xylene Substitute Mountant (Thermo Fisher Scientific Inc., PA). Picrosirius Red/Fast Green staining solution was prepared by dissolving 0.1% direct Red 80 and 0.1% Fast Green FCF in saturated aqueous picric acid (1.2% picric acid in water, Sigma Aldrich, MO). Picrosirius Red/Fast Green solution was then applied to the sections and sections were incubated for 1 h.^{[53 (link)]} Samples were rinsed with DI water, dehydrated with alcohol, cleaned with xylene, and then mounted. Finally, samples were imaged using a BX51 microscope (Olympus, Japan).

MSC and chondrogenic spheroids were fixed with 4% paraformaldehyde and sectioned with paraffin embedding to obtain 10 μm sections. H&E staining was performed on the sections using Leica Autostainer XL (Leica, Germany). For sGAG visualization using Toluidine Blue O staining, sections were incubated in a Toluidine Blue solution (0.1% in DI water, Sigma Aldrich, MO) at room temperature for 2 min. The dye was then removed and samples were washed twice with DI water, followed by dehydration with ascending alcohol and clearing with xylene. All samples were mounted and imaged using the EVOS microscope.