C7974

Constructs were fixed in 4% paraformaldehyde with 100 mM sodium cacodylate overnight. Subsequently, constructs were dehydrated through a series of ethanol washes, cleared in xylenes, embedded in paraffin, and sectioned at 8 μm per section. Sections from each sample were stained for GAGs with 0.1% aqueous Safranin-O, collagen with 0.02% aqueous fast green, and stained for cell nuclei with hematoxylin. Immunohistochemistry was performed with monoclonal antibodies to type I collagen (ab90395; Abcam, Cambridge, MA), type II collagen (II-II6B3; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or type X collagen (C7974; Sigma-Aldrich). For all IHC, slides with sections of each sample were cleared, rehydrated, and digested with pepsin for epitope retrieval (Digest-All, Invitrogen). After probing with appropriate primary antibodies, all samples were probed with the same biotinylated anti-mouse secondary antibody (ab97021; Abcam), treated with HRP conjugate (Invitrogen), and finally incubated with chromagenaminoethylcarbazole (AEC) single solution (Invitrogen). Human osteochondral plugs were used as positive controls for each antibody and for the general histological stain. Negative controls without primary antibody were also prepared for each slide.

Representative pellets (n=2) were fixed in 4% paraformaldehyde at 4°C overnight. After embedding in paraffin blocks, 5-μm sections were stained for GAG with Alcian blue (Sigma-Aldrich, counterstained with fast red). For immunostaining, the sections were immunolabeled with primary antibodies against type II collagen (dilution 1:100, catalog number II-II6B3; Developmental Studies Hybridoma Bank, Iowa City, IA) and type X collagen (clone COL-10, dilution 1:1000, catalog number C7974, Sigma-Aldrich), followed by the secondary antibody. Immunoactivity was detected using Vectastain ABC reagent (Vector, Burlingame, CA) with 3, 3’-diaminobenzidine as a substrate.

Prior to fixation, all constructs in osteogenic media and three chondrogenic negative controls were decalcified in 2 mL of 5% formic acid in deionized water for 30 min at room temperature. Decal solution was collected and frozen at −20°C for calcium quantification. Constructs (n=2/group) were fixed, dehydrated, cleared, paraffin embedded, and sectioned (8 μm thickness) as described previously [49 (link)]. Immunohistochemistry (IHC) was performed with monoclonal antibodies against type I collagen (ab90395; Abcam, Cambridge, MA), type II collagen (II-II6B3; Developmental Studies Hybridoma Bank, University of Iowa), or type X collagen (C7974; Sigma-Aldrich) using aminoethyl carbazole (Invitrogen) as the chromogen as described previously [49 (link)]. Xylene-cleared sections were also histologically stained using 0.1% aqueous Safranin-O, 0.02% fast-green, and hematoxylin. Human osteochondral tissue was used as a positive control. Negative controls for IHC did not include primary antibody. Sections were imaged on a CKX41 scope with a DP26 camera (Olympus).