Spheroids were fixed with 10% formalin neutral buffer solution (Wako), dehydrated, and
embedded in paraffin. Specimens were cut into 4-µm sections. Safranin O
staining was performed for the detection of proteoglycans. For immunohistochemistry of
type II, I, and X collagen, sections were treated with 50
µg/ml proteinase K (Promega, Madison, WI, U.S.A.) for
10 min at room temperature. For type II collagen, an additional antigen retrieval step was
carried out using 25 mg/ml hyaluronidase (Sigma) for 2 hr at 37°C.
Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30
min. After washing with Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the
sections were blocked with TBS-T containing 10% normal goat serum (Sigma) for 30 min at
room temperature, and then incubated with rabbit anti-type II collagen antibody (1:200;
LSL, Tokyo, Japan), mouse anti-type I collagen antibody (1:1,000; Abcam, Cambridge, U.K.),
and mouse anti-type X collagen antibody (1:1,000; Sigma) at 4°C overnight. The slides were
washed with TBS-T and incubated with horseradish peroxidase-labeled polymer (Dako, Tokyo,
Japan) for 1 hr at room temperature. Finally, diaminobenzidine substrate (Dako) was placed
on the slides and all slides were counterstained with hematoxylin.
embedded in paraffin. Specimens were cut into 4-µm sections. Safranin O
staining was performed for the detection of proteoglycans. For immunohistochemistry of
type II, I, and X collagen, sections were treated with 50
µg/ml proteinase K (Promega, Madison, WI, U.S.A.) for
10 min at room temperature. For type II collagen, an additional antigen retrieval step was
carried out using 25 mg/ml hyaluronidase (Sigma) for 2 hr at 37°C.
Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30
min. After washing with Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the
sections were blocked with TBS-T containing 10% normal goat serum (Sigma) for 30 min at
room temperature, and then incubated with rabbit anti-type II collagen antibody (1:200;
LSL, Tokyo, Japan), mouse anti-type I collagen antibody (1:1,000; Abcam, Cambridge, U.K.),
and mouse anti-type X collagen antibody (1:1,000; Sigma) at 4°C overnight. The slides were
washed with TBS-T and incubated with horseradish peroxidase-labeled polymer (Dako, Tokyo,
Japan) for 1 hr at room temperature. Finally, diaminobenzidine substrate (Dako) was placed
on the slides and all slides were counterstained with hematoxylin.