Fn antibody

For immunofluorescence, plates were fixed in 4% paraformaldehyde and stained with polyclonal FN antibody (Sigma, Cat#3648, 1:200 dilution). If not decellularized, cells were also stained with DAPI, but were never permeabilized during staining. In studies involving labeling with Aha, cells were cultured for two days in glutamine-, methionine- and cystine-free high-glucose DMEM (Gibco, Waltham, MA, USA) containing 1% penicillin/streptomycin (Gibco), 1% glutagro (Corning, Corning, NY, USA), 0.2 mM cystine (Sigma), and 40 µg/mL of either Aha (Click Chemistry Tools, Scottsdale, AZ, USA) or Met (Sigma) as a control. For nascent protein visualization, cells were incubated in fresh media without Aha or methionine, but with 30 μM Alexa Fluor DBCO-647 (Click Chemistry Tools) for 30 min at 37  °C. Following incubation, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature [38 (link),39 (link)].

Sections were deparaffinized in xylene, hydrated in graded ethanol solutions, and hematoxylin and eosin (H&E) staining were performed as per published protocols37 (link)38 (link). Alizarin red staining to visualize calcium deposition was performed as per standard procedures. Immunohistochemistry using peroxidase conjugated secondary antibodies or fluorescent probes according to published protocols. The following antibodies were used, rabbit anti-fibronectin (FN) antibody (1/100, Sigma), mouse-runt-related transcription factor 2 (RUNX2) antibody (1/100, Abcam), mouse osteocalcin (OCN) antibody (1/1000, Abcam) and mouse EIF3β (TRIP-1) antibody (1/500 Santacruz Biotechnology). All fluorescently stained sections were imaged at the University of Illinois at Chicago Research Resource Center core imaging facility. Imaging was performed using a Zeiss LSM 710 confocal microscope equipped with Zen image analysis software. All comparative fluorescence images were obtained using the same imaging conditions.