Table 1

Details of the immunohistochemistry methodology employed

Target

Antigen retrieval

Blocking step and primary antibody

Secondary antibody

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)

Le Maitre et al. Arthritis Research & Therapy 2009 11:R20   doi:10.1186/ar2611