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.