Alcian blue stain

Adipogenic, osteogenic and chondrogenic induction of PDMSCs was performed according to manufacturer’s instructions in the StemPro® Adipogenesis, Osteogenesis, and Chondrogenesis Differentiation Kit (Gibco). PDMSCs at passage 2 were seeded on polystyrene surfaces (control) and cultured in low-glucose DMEM-10% FBS for 3 days prior to induction of differentiation. For adipogenic induction, PDMSCs were cultured on StemPro® Adipocyte Differentiation Basal Medium supplemented with StemPro® adipogenesis supplement and gentamycin (10 mg/mL, Gibco) for 7 days. Adipogenesis was assessed by Oil Red O staining (Sigma-Aldrich, MO, USA). For induction of chondrogenic differentiation, PDMSCs were cultured in StemPro® Chondrocyte Differentiation Basal Medium with StemPro® chondrogenesis supplement and gentamycin reagent (10 mg/mL) for 12 days. Chondrogenesis was assessed with 1% Alcian blue stain (Sigma-Aldrich) prepared in 0.1 N HCL. For induction of osteogenic differentiation, PDMSCs were cultured in StemPro® Osteocyte Differentiation Basal Medium with StemPro® osteogenesis supplement and gentamycin reagent (10 mg/mL) for 13 days. Osteogenesis was assessed by Alizarin Red S staining (Sigma-Aldrich). Cells were fixed prior to staining. Images were captured with an inverted light microscope (Leica Microsystems, IL, USA).

5 μm sections routinely cut from formalin fixed, paraffin wax-embedded (FFPE) tissue sections were deparaffinised in xylene (Sigma) for 10 min, re-hydrated in decreasing concentrations of ethanol (Sigma) and finally soaked in tap water. For collagen I and III staining, slides were incubated in Picrosirus red stain (Abcam) for 1 hr and then washed in 0.5% acetic acid (Sigma) for stain differentiation. For GAG staining, slides were incubated in Alcian blue stain (Sigma) for 20 min^{57 (link)} and counterstained with eosin (Dako UK Ltd., Eli, UK). Slides were then dehydrated in increasing concentrations of ethanol, cleared in two changes of xylene and finally mounted with DPX mounting medium (Sigma).

Calcium and proteoglycan content of mixed populations of ATDC5-ctrl, ATDC5-wtTUFT1, and ATDC5-mutTUFT1 cells were determined by staining the cell layers with Alizarin red stain (Sigma, St Louis, USA) or Alcian blue stain (Sigma, St Louis, USA), respectively. Cells were grown 15 days after their mineralization state was analyzed. To evaluate calcium concentration, ATDC5 cells were fixed with 4% paraformaldehyde (PFA) for 5 min at 4°C, washed with PBS, stained with 2% Alizarin red (pH 4.2) for 5 min in room temperature, washed with distilled water and bound dye was extracted with 10% cetylpyridium chloride for 10 min. Optical density (OD) of the samples was determined at 570 nm by spectrophotometry. Proteoglycan content was analyzed by washing ATDC5 cells with PBS, fixing with 95% methanol for 20 min, staining with 1% Alcian blue 8GX (Sigma, St Louis, USA) in 0.1M HCl overnight and rinsing with distilled water. Cell cultures were extracted with 6M guanidine-HCl for 6h at room temperature and the released dye was evaluated by measuring the OD at 630 nm by spectrophotometer.