Advance hrp enzyme

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.

Pellets aggregates were dehydrated, embedded in paraffin and sectioned after being fixed in 4% paraformaldehyde (all Sigma) as described previously [44 (link)]. Alcian Blue (Sigma) stainings for detection of proteoglycans in pellet sections were performed as outlined in our earlier research [44 (link)]. Further, immunohistochemical stainings were performed using following compilation of antibodies and pre-digestion: collagen type II (COL II; encoded by COL2A1)-pepsin (1 mg/ml; Sigma)/monoclonal anti-COL II antibodies (Acris Antibodies GmbH, Hiddenhausen, Germany) and COL X—0.25% trypsin (Sigma)/polyclonal anti-COL X antibodies (Calbiochem, Bad Soden, Germany). Diaminobenzidine staining (DAB Kit; Sigma) following treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) was used to visualize immunohistochemical stainings. Sections were subsequently counterstained with hemalaun (Merck, Darmstadt, Germany). Non-immune IgGs (Sigma) replacing primary antibodies were used as negative controls. For detailed information regarding antibodies and immunohistochemistry please refer to our previous work [45 (link), 49 (link), 51 (link)].

Type I collagen hydrogel aggregates were fixed at day 21 in 4% paraformaldehyde, followed by dehydration, paraffin embedding, sectioning and staining with hematoxylin/eosin (H&E), alcian blue and ALP (all Sigma) according to previously published protocols [33 (link)]. Alternate sections were used for immunohistochemistry. The following antibody treatments were used: type II collagen (COL II) (pepsin (1 mg/ml; Sigma)/monoclonal anti-COL II antibodies (Acris Antibodies GmbH, Hiddenhausen, Germany)); chondroitin-4-sulfate (CS4) (chondroitinase ABC (5 U/mL; Sigma)/monoclonal anti-CS4 antibodies (Millipore GmbH, Schwalbach, Germany)); type X collagen (COL X) (0.25% trypsin; Sigma)/polyclonal anti-COL X antibodies (Calbiochem, Bad Soden, Germany)). Visualization of the immunostainings was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma). Thereafter, the slides were counterstained with hemalaun (Merck, Darmstadt, Germany). Controls with non-immune IgG (Sigma) were performed for all immunohistochemical analyzes.