Tissues (neo-cartilage and neo-bone) were fixed in 4% formaldehyde and embedded in paraffin. After sectioning, slides were deparaffinized and rehydrated prior to histology or immunohistochemistry.
Overall cellular and tissue structure was visualized with hematoxylin–eosin (HE) staining. Glycosaminoglycans were visualized by staining with 1% Alcian Blue 8-GX (Sigma-Aldrich) and Nuclear Fast red staining (Sigma-Aldrich). Calcium deposits were stained with 2% Alizarin Red S (Sigma-Aldrich).
To detect COL2 (MAB1330; Millipore; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), COL1 (ab34710; Abcam; 1:1000 in TBST/10% normal goat serum, overnight at 4 °C), and COL10 (× 53/2031501005; Quartett; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), immunohistochemistry was performed with 3-diaminobenzidine (DAB) solution (Sigma-Aldrich) and hematoxylin (Klinipath) as described before (Bomer et al. 2015 (link)).
Lipid droplets were stained for 10 min with Oil-Red-O solution (Sigma-Aldrich) after fixation of the cells in 4% formaldehyde. To reduce the background, the following staining cells were gently washed with 60% isopropanol and distilled water.
Overall cellular and tissue structure was visualized with hematoxylin–eosin (HE) staining. Glycosaminoglycans were visualized by staining with 1% Alcian Blue 8-GX (Sigma-Aldrich) and Nuclear Fast red staining (Sigma-Aldrich). Calcium deposits were stained with 2% Alizarin Red S (Sigma-Aldrich).
To detect COL2 (MAB1330; Millipore; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), COL1 (ab34710; Abcam; 1:1000 in TBST/10% normal goat serum, overnight at 4 °C), and COL10 (× 53/2031501005; Quartett; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), immunohistochemistry was performed with 3-diaminobenzidine (DAB) solution (Sigma-Aldrich) and hematoxylin (Klinipath) as described before (Bomer et al. 2015 (link)).
Lipid droplets were stained for 10 min with Oil-Red-O solution (Sigma-Aldrich) after fixation of the cells in 4% formaldehyde. To reduce the background, the following staining cells were gently washed with 60% isopropanol and distilled water.
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