Ab746p

Immunohistochemistry was performed as previously described (Warnock et al., 2012 (link)) for type I collagen (AB749P; 1:100 dilution; Millipore), type II collagen (AB746P; 1:100: Millipore), macrophage MAC387 receptor to determine type A synoviocyte content, (CBL260; 1:200 dilution; Millipore); and alpha smooth muscle actin (M0851; 1:30; Dako). Extracellular and intracellular immunoreactivity intensity and prevalence was scored as previously described (Wakshlag et al., 2011 (link)) with some modifications: immunoreactivity was localized to intracellular or extracellular staining, and ECM immunoreactivity intensity was described and scored, as negative (0), mild (1), moderate (2), or strong (3) staining. As determined by hand count, intracellular immunoreactivity and extracellular immunoreactivity was categorized as positive in <10%, 10–50%, or >50% of cells and sample area, respectively. Each of these histologic observations was assigned a score (Table 2). Then a histologic intensity coefficient was calculated for each ECM component, as follows: [[(Extracellular matrix staining intensity score) × (percentage area coverage of positive staining score)] + [(Intracellular staining intensity score) × (percentage positive staining cells score)]]/2 (Table 1).

The Prestwick Chemical Library was purchased from Prestwick Chemical (Illkirch-Graffenstaden, France) and supplied in a special academic format with the 1280 FDA-approved compounds at 10 mM concentration in DMSO, in 16 96-well format plates, each containing 80 compounds.
Resveratrol (CAS no. 501-36-0, Enzo Life Sciences, Farmingdale, NY, USA) and dipyridamole (CAS no. 58-32-2, Sigma, Buchs, SG, Switzerland) were dissolved in DMSO and prepared as 1 M and 200 mM stock solutions, respectively.
Recombinant human BMP2 [rhBMP2; Chinese hamster ovary (CHO)-derived, R&D Systems, Minneapolis, MN, USA] was prepared as a 100 µg/ml stock solution in 4 mM HCl containing 0.1% bovine serum albumin (BSA; Sigma-Aldrich, Buchs, SG, Switzerland).
Antibodies for western blot analyses were: anti-p-Smad1/5 (13820S, Cell Signaling, Danvers, MA, USA), anti-GAPDH (MAB3749, Millipore, Billerica, MA, USA), and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies (Dako, Glostrup, Denmark). For immunohistochemical analyses, the following antibodies were used: rabbit anti-bovine polyclonal antibody anti-collagen type II (AB746P, Millipore, Billerica, MA, USA), rabbit polyclonal antibody anti-Sox9 (AB5535, Millipore, Billerica, MA, USA), and anti-rabbit (K4002) and anti-mouse (K4000) EnVision System-HRP Labelled Polymer (Dako, Glostrup, Denmark).