Image studio software version 3.1.4

For detection of COLI and phosphorylation of SMAD 1/5/8 (Ser 463/Ser 465) (p-SMAD1/5/8) cells were fixed with 70% ethanol after 24 h and 4 days of culture in osteogenic medium. After being rinsed with PBS, the cells were incubated with 10% bovine serum albumin for 60 min at room temperature before being incubated overnight with COLI (sc-8784-R, Santa Cruz Biotechnology, Inc.) or p-SMAD1/5/8 (sc-12353-R, Santa Cruz Biotechnology, Inc.), dilution 1:100, 4°C. After being rinsed with PBS, the cells were incubated for 60 min with a biotinylated antibody (E0432, DAKO), dilution 1:300. They were again rinsed with PBS, then incubated 120 min with streptavidin and Alexa Fluor® 700 conjugate (S-21383, Life Technologies) for detection of COLI or streptavidin and Alexa Fluor^® 488 conjugate (S-11223, Life Technologies) for detection of p-SMAD1/5/8, dilutions 1:500. After being rinsed with PBS, the cells were dried overnight at 4°C. Quantitative detection of COLI was then performed using Odyssey^® Sa Infrared Imaging (Licor Biosciences) and Image Studio software (version 3.1.4) (Licor Biosciences). Cells for p-SMAD1/5/8 evaluation were mounted in glycergel mounting medium and visualized by fluorescence microscopy.