hPDLSCs placed on CTRL and TEST samples were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. After PBS wash, cultures were made for immunofluorescence labelling. Then, cells seeded on granules were permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 5% skimmed milk in PBS. Primary monoclonal antibodies to anti-human Fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Laminin (Santa Cruz Biotechnology), N-Cadherin (Santa Cruz Biotechnology) and RUNX2 (Santa Cruz Biotechnology) were utilized, followed by Alexa Fluor 488 green fluorescence conjugated goat anti-mouse as secondary antibodies (Molecular Probes, Invitrogen, Eugene, OR, USA). Then, the specimens were incubated with Alexa Fluor 594 phalloidin red fluorescence conjugate (Molecular Probes) to stain actin cytoskeleton. Nuclei were dyed with TOPRO (Molecular Probes). Specimens were positioned facing down on glass slides and mounted with Prolong antifade (Molecular Probes) [27 (link)]. The stained samples were evaluated using a Zeiss LSM800 META confocal system, connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA). The images were taken using an argon laser beam with excitation lines at 488 nm.
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