Aec red single histostain plus bs

Pellets were fixed in 10% formalin overnight, dehydrated to 70% ethanol, then embedded in paraffin and sectioned (8 μm). For immunohistochemistry of type II collagen (1:1 Iowa Hybridoma Bank, IA), sections were deparaffinized with xylenes, rehydrated with decreasing ethanol concentrations, incubated with Digest-All three pepsin digestion (Thermo Fisher) for 5 min, treated with 30% peroxidase, blocked with 10% goat serum, incubated with primary antibody for 1 hr at room temperature, incubated with secondary antibody (1:500, Abcam, ab9702) for 30 min, treated with HRP streptavidin and AEC Red Single (Histostain-Plus BS, Thermo Fisher), counterstained with hematoxylin, and mounted with Vectamount (Thermo Fisher).

To perform immunohistochemistry of type II collagen (1:1, Iowa Hybridoma
Bank, Iowa), type VI collagen (1:200, Fitzgerald, Cambridge UK), or type I
collagen (1:800, Abcam, Cambridge MA), sections were treated with xylenes and a
decreasing ethanol series, incubated for 5 min with Digest-All 3 pepsin
digestion (Thermo Fisher), treated with 30% peroxidase, blocked with goat serum,
incubated with primary antibody for 1 h at room temperature, incubated with
secondary antibody (ab97021 for type II and type I collagen, ab6720 for type VI
collagen, Abcam, 1:500) for 30 minutes, treated with HRP streptavidin and AEC
Red Single (Histostain-Plus BS, Thermo Fisher), counterstained with hematoxylin,
and mounted with Clearmount (Thermo Fisher). Human osteochondral sections were
used as both positive and negative controls.