Aggregates were harvested on d14 and d28, fixed in 4% paraformaldehyde and 10μm thick frozen sections were prepared.
Sections were stained with dimethylmethylene blue (DMMB) (Sigma Aldrich) for glycosaminoglycans. Histochemical ALP staining was performed with an alkaline phosphatase kit (Sigma Aldrich) with neutral red as counterstain.
For immunohistochemistry mouse anti collagen type X (1:20, Quartett Immunodiagnostika und Biotechnologie GmbH) and mouse anti collagen type II (1:100, Calbiochem) antibodies were used and immunohistochemistry was carried out as follows: . After blocking of endogenous peptidases (3% H2O2/ 10% Methanol in PBS) for 30 minutes, antigen retrieval with pepsin digestion for 15 minutes at room temperature (RT) was performed. Then sections were incubated in blocking buffer (10% fetal bovine serum/10% goat serum in PBS) for 60 minutes at RT followed by incubation in an appropriate primary antibody in blocking buffer overnight at 4°C. For collagen type X staining additional hyaluronidase digestion for 60 minutes at RT was performed prior to blocking. Immunolabeling was detected with a biotinylated secondary antibody (1:100; Dianova), horseradish peroxidase conjugated streptavidin (Vector Laboratories, Burlingame) and metal enhanced diaminobenzidine as substrate (Sigma Aldrich).
Sections were stained with dimethylmethylene blue (DMMB) (Sigma Aldrich) for glycosaminoglycans. Histochemical ALP staining was performed with an alkaline phosphatase kit (Sigma Aldrich) with neutral red as counterstain.
For immunohistochemistry mouse anti collagen type X (1:20, Quartett Immunodiagnostika und Biotechnologie GmbH) and mouse anti collagen type II (1:100, Calbiochem) antibodies were used and immunohistochemistry was carried out as follows: . After blocking of endogenous peptidases (3% H2O2/ 10% Methanol in PBS) for 30 minutes, antigen retrieval with pepsin digestion for 15 minutes at room temperature (RT) was performed. Then sections were incubated in blocking buffer (10% fetal bovine serum/10% goat serum in PBS) for 60 minutes at RT followed by incubation in an appropriate primary antibody in blocking buffer overnight at 4°C. For collagen type X staining additional hyaluronidase digestion for 60 minutes at RT was performed prior to blocking. Immunolabeling was detected with a biotinylated secondary antibody (1:100; Dianova), horseradish peroxidase conjugated streptavidin (Vector Laboratories, Burlingame) and metal enhanced diaminobenzidine as substrate (Sigma Aldrich).