K4002

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).

Paraffin-embedded kidney tissues were cut into 5-µm sections and deparaffinised. Sections were permeabilised with PBST for 3 days at 4 °C and incubated with primary antibodies against DsRed2 overnight at 4 °C. Next, sections were incubated with the Envision + System horseradish peroxidase-labelled polymer anti-rabbit IgG (K4002, Dako, Glostrup, Denmark) for 30 min at room temperature (RT), then visualised with 0.01% DAB containing 1% nickel in 0.05 M Tris-HCl buffer (pH 7.6) for 60 min at RT. Sections were also stained with H&E and observed under light microscopy (NIS element BR 3.0; Nikon).