Adhesion of hPDLSCs on Granules

To better understand and evaluate the adhesion on G, the hPDLSCs on granules were processed for immunofluorescence labeling. Samples were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4. Then, samples were permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 5% skimmed milk in PBS. Primary monoclonal antibodies to antivinculin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-VEGF (1:250; Santa Cruz Biotechnology) were used, followed by Alexa Fluor 568 green fluorescence conjugated goat antimouse as secondary antibodies (Molecular Probes, Invitrogen, Eugene, OR, USA). Subsequently, samples were incubated with Alexa Fluor 488 phalloidin green-fluorescence conjugate (Molecular Probe) as cytoskeleton actin marker. Samples were placed face-down on glass slides and mounted with Prolong antifade (Molecular Probes) [49 (link)]. Samples were observed with CLSM (Zeiss LSM510META) and connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA).