Anti smo antibody

The pharmacological reagent cyclopamine was provided by Selleck Chemicals (Houston, TX, USA). Recombinant human Gal-1 (rGal-1) was obtained from Peprotech (Rocky Hill, NJ, USA), and dissolved in 0.1% bovine serum albumin (BSA; Biosharp, Anhui, China). Anti-galectin-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SMO antibody (Abcam, Cambridge, UK), anti-Gli-1 antibody (Abcam), anti-SHH antibody (Abcam), anti-E-Cadherin antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Vimentin (Cell Signaling Technology), anti-β-actin antibody (Beyotime, Jiangsu, China), HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) were used in this study.

OA-MSCs and OACs were collected and treated with 1 × radioimmuno-precipitation lysis buffer (Thermo Fisher Scientific) supplemented with Halt protease inhibitor (Cat#78430, Thermo Fisher Scientific). Cell lysis was then sonicated for 15 s, heated to 95°C for 5 min, and stored at −20°C until use for the sodium dodecyl sulfatepoly acrylamide gel electrophoresis; 20-μg protein samples were loaded and transferred to nitrocellulose membrane probed with anti-SHH antibody (Cat#2207, at 1:1,000 dilution, Cell Signaling Technology, Danvers, MA, United States), anti-SMO antibody (Cat#ab235183, at 1:500 dilution, Cambridge, MA, United States), anti-RUNX2 antibody (Cat# ab23981, at 1:5,000 dilution, Abcam), anti-SOX9 (Cat# ab185230, at 1:2,000 dilution, Abcam), and anti-MMP13 antibody (Cat#39012, at 1:3,000 dilution, Abcam) and normalized with β-Actin (Cat#ab108349, at 1:1,000 dilution, Abcam, United States). After the primary antibody incubation, the membranes were labeled with secondary anti-rabbit IgG antibody (at a dilution of 1:15,000, IRDye infrared dye, LI-COR Biosciences, Lincoln, NE, United States) and were then imaged using the Licor Odyssey CLx scanner (LI-COR Biosciences). Quantification of Western blot data by evaluating the gray values of protein expression level was performed using software in the Odyssey Infrared Imaging system.

The cassettes with OA cartilage were sectioned at 6-μm thickness, mounted onto slides, heated at 58°C for 30 min, cleared with xylene twice, and rehydrated using sequential incubation in 100, 95, and 70% ethanol for 10 min, respectively. Antigen retrieval was performed by treating samples with HA and H₂O₂. After blocking with 10% goat serum, slides were incubated with the primary antibodies: anti-SHH (Cat#06-1106, 1:100, Millipore, Sigma), anti-SMO antibody (1:100, Abcam), and mouse anti-CD166 (Cat#MABN1785, 1:100, EMD Millipore) in antibody dilution buffer overnight at 4°C. Slides were then incubated with the secondary antibodies: goat anti-rabbit IgG Alexa Fluor 594 (Cat#ab150080, 1:200, Life Technologies) and donkey anti-mouse IgG H&L Alexa Fluor 488 (Cat#ab150105, 1:200, Abcam) in for 30 min. Finally, slides were mounted using anti-fade mounting medium with 4′-6-diamidino-2-phenylindole (DAPI). Negative control was prepared only with secondary antibodies (data not shown). Images were captured by using Nikon Eclipse 90i and NIS elements imaging systems, and then processed by Image J (Laboratory for Optical and Computational Instrumentation, Madison, WI, United States).