Histological and Immunohistochemical Assessment
Representative constructs were harvested and fixed overnight in 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.2), processed in graded solvents and embedded in paraffin. Thin (5 µm) sections were cut and mounted on Superfrost™ slides (Fisher Scientific) and dried for 24 hours at 37°C. Sections were stained with safranin-O (proteoglycans) and sirius red (collagen) or assessed for localization of collagen types I and II by immunohistochemistry, as described previously (Khan et al. , 2009). Briefly, sections were deparaffinized, rehydrated, and then treated for antigen retrieval. For collagen I, sections were heated in citrate buffer (10 mM citric acid, 0.05% Tween 20; pH 6) at 95-100oC for 20 minutes. For collagen II, sections were treated with 0.25 units/mL chondroitinase ABC (1 hour) and 0.25 units/mL keratinase (30 minutes) in tris-acetate buffer (40 mM tris acetate, 1 mM EDTA; pH 8.5) at 37°C. Sections were then blocked with 1% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature and incubated with antibodies against collagen I (Abcam ab90395, 1:100 dilution) or collagen II (Developmental Studies Hybridoma Bank II-II6B3, 1:100 dilution) in 1% BSA in PBS, at 4°C overnight. Sections were then incubated with Texas Red-labelled goat anti-mouse secondary antibody (Abcam ab6787, 1:200 dilution in 1% BSA in PBS) for 2 hours at room temperature. Sections were counterstained with DAPI mounting medium (Vector Laboratories), and imaged (ZOE Fluorescent Cell Imager). Negative controls were performed by replacing primary antibodies with 1% BSA in PBS, with no staining detected.