Anti igfbp7

ASCs were lysed at day 0, 1, 2, 3, 4, 5, 6 or 7 following the differentiation and non-differentiation protocols and proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-α-SMA (1:10,000, rabbit, monoclonal, cat# 04-1094, Millipore, Billerica, MA, USA), anti-SM22α (1:500, mouse, monoclonal, cat# 28811, Abcam, Cambridge, UK), anti-caldesmon (1:500, mouse, monoclonal, cat# MAB3576, Millipore, Billerica, MA, USA), anti-IGFBP7 (1:300, goat, polyclonal, cat# AF1334, R&D Systems, Minneapolis, MN, USA). Washed membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000‒1:50,000, cat# A9044, cat# AP106P, cat# AP187P Sigma-Aldrich, St. Louis, MS, USA and Millipore, Billerica, MA, USA). Immunoreactive proteins were detected by chemiluminescence. Signals were revealed by chemiluminescence, visualized by autoradiography and quantified densitometrically. β-Actin (1:50,000, mouse, monoclonal, cat# A2228, Sigma-Aldrich, St. Louis, MO, USA) expression was used as a housekeeping protein.

Immunofluorescence was performed on methanol-fixed cells initially blocked with 10% normal goat serum in PBS supplemented with 0.1% Triton-X for 1 h at RT. Slides were incubated with the following primary antibodies: anti-α-SMA (1:100, rabbit, monoclonal, cat# 04-1094, Millipore, Billerica, MA, USA), anti-SM22α (1:50, mouse, monoclonal, cat# 28811, Abcam, Cambridge, UK), anti-caldesmon (1:50, mouse, monoclonal, cat# MAB3576, Millipore, Billerica, MA, USA), anti-IGFBP7 (1:100, goat, polyclonal, cat# AF1334, R&D Systems, Minneapolis, MN, USA). After washing three times in PBS, slides were incubated with Alexa 568 IgG (1:300, goat, polyclonal, cat# A-11004, Thermo Fisher Scientific, Rockford, IL, USA). Glass slides were then rinsed in PBS and milli-Q water and coverslipped in fluorescent mounting medium (Dako Diagnostics, Glostrup, Denmark) spiked with Hoechst (DAPI, 1:1000, cat# D9542, Sigma-Aldrich, St. Louis, MO, USA). Sections were visualized under the Olympus 1 × 2 UCB fluorescent microscope and images were captured using in vivo v.3.2.2 software (MediaCybernetics, Rockville, MD, USA).