Aggrecan |
0.1% w/v hyaluronidase in Tris-buffered saline (Sigma, Poole, UK), 30 minutes at 37°C |
20% v/v rabbit serum, and mouse monoclonal aggrecan 1° (1:25 dilution; AbCam, Cambridge, UK) |
Biotinylated rabbit anti-mouse antiserum (1:400; Dako, Ely, Cambridgeshire, UK) |
Sox-9 |
None required |
20% v/v swine serum, and rabbit polyclonal Sox-9 1° (1:100 dilution; SantaCruz, Heidelburg, Germany) |
Biotinylated swine anti-rabbit antiserum (1:400; SantaCruz) |
Type I collagen |
0.01% hyaluronidase (Sigma), 0.02% trypsin (Sigma) w/v in Tris-buffered saline |
20% v/v rabbit serum, and mouse monoclonal type I collagen 1° (1:250 dilution; ICN, Basingstoke, UK), |
Biotinylated rabbit anti-mouse antiserum (1:400; Dako) |
Type II collagen |
0.1% w/v hyaluronidase in Tris-buffered saline (Sigma), 30 minutes at 37°C |
20% v/v rabbit serum, and mouse monoclonal type II collagen 1° (1:100 dilution; MP Biomedicals, Illkirch, France) |
Biotinylated rabbit anti-mouse antiserum (1:400; Dako) |
Type X collagen |
1 mg/ml hyaluronidase, 0.25 U/ml chondrotinase in Tris-buffered saline, 1 hour at 37°C; followed by 0.1% protease in Tris-buffered saline, 10 minutes at 37°C |
25% v/v goat serum, and mouse monoclonal type X collagen 1° (1:200 dilution; AbCam) |
Biotinylated goat anti-mouse antiserum (1:100; SantaCruz) |
Caspase 3 |
None required |
20% v/v donkey serum, and goat polyclonal caspase 3 1° (1:500 dilution; SantaCruz) |
Biotinylated donkey anti-goat antiserum (1:300; SantaCruz) |