*4.7. Histochemical Staining*

On days 7, 14 and 21 of osteogenic, chondrogenic and adipogenic differentiation, the cells were washed with PBS and fixed with 4% paraformaldehyde (POCH, Avantor Performance Materials Poland S.A., Gliwice, Poland) for 30 min at RT.

To evaluate calcium phosphate deposition in the cells differentiated into osteoblasts, the cells were rinsed twice with distilled water following fixation and stained with 2% Alizarin Red S solution with pH 4.2 (Sigma-Aldrich) for 3 min.

To visualize sulphated proteoglycans that are characteristic for chondrogenic differentiation, fixed cells were rinsed with PBS and stained with 1% Alcian Blue solution (Sigma-Aldrichprepared in 0.1 N HCl (POCH, Avantor Performance Materials Poland S.A) for 30 min. The wells were rinsed thrice with 0.1 N HCl (POCH) and then distilled water was added to neutralize the acidity.

To visualize the presence of oil droplets during adipogenic differentiation, the cells were rinsed with distilled water and incubated with 60% isopropanol (POCH, Avantor Performance Materials Poland S.A) for 5 min. After the removal of isopropanol, the cells were stained with 1% Oil Red O solution (Sigma-Aldrich) for 5 min.

After histochemical staining, the cells were rinsed with distilled water and visualized using an Olympus IX81 microscope equipped with MicroPublisher 3.3 RTV camera (Olympus, Tokyo, Japan).
