For histology, aggregates were fixed in 4% paraformaldehyde for 1 h, followed by dehydration in graded alcohols, paraffin embedding, sectioning to 4 μm, and staining with haematoxylin/eosin (H&E) or alcian blue (Sigma) as described previously [7 (link), 8 (link)]. For visualisation of ALP activity, a histochemical assay was performed according to the instructions of the supplier (Sigma).
Immunohistochemistry on alternate sections was performed as described previously [7 (link)]. Briefly, following the respective pre-treatments with pepsin (1 mg/mL), or chondroitinase ABC (Sigma; 5 U/mL), or trypsin (0.25%) sections were incubated overnight with the following primary antibodies: monoclonal anti-COL type II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-chondroitin-4-sulphate (CS4) (Millipore GmbH, Schwalbach, Germany) or anti-collagen type X (COL type X) (Calbiochem, Bad Soden, Germany). Immunohistodetection was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma), and slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). In addition, controls with non-immune Ig G (Sigma) instead of the primary antibodies were also performed.
Immunohistochemistry on alternate sections was performed as described previously [7 (link)]. Briefly, following the respective pre-treatments with pepsin (1 mg/mL), or chondroitinase ABC (Sigma; 5 U/mL), or trypsin (0.25%) sections were incubated overnight with the following primary antibodies: monoclonal anti-COL type II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-chondroitin-4-sulphate (CS4) (Millipore GmbH, Schwalbach, Germany) or anti-collagen type X (COL type X) (Calbiochem, Bad Soden, Germany). Immunohistodetection was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma), and slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). In addition, controls with non-immune Ig G (Sigma) instead of the primary antibodies were also performed.
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