Immunofluorescent Analysis of hPDLSCs

hPDLSCs and hPDLSCs/LPS-G were processed as previously reported by Trubiani et al.^{35 (link)} The following primary monoclonal antibodies were used: antihuman NF-kB (1:250, rabbit) (OriGene Technologies, Inc., Rockville, MD, USA), anti-DNMT1 (1:250, rabbit) (OriGene), anti-p300 (1:250, rabbit) (OriGene). Then, cells were incubated for 1 h at 37°C with Alexa Fluor 568 red fluorescence conjugated goat anti-rabbit secondary antibodies (1:200) (Molecular Probes). Subsequently, cells were incubated with Alexa Fluor 488 phalloidin green fluorescence conjugate (1:200, Molecular Probes) to mark cytoskeleton actin. Cell nuclei were stained with TOPRO (1:200, Molecular Probes) for 1 h at 37°C. Glass coverslips were placed upside down on glass slides and mounted with Prolong antifade (Molecular Probes).^{36 (link)} Samples were observed with Zeiss LSM510META confocal system (Zeiss, Jena, Germany) connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (63x). Images were collected using an argon laser beam with excitation lines at 488 nm and a helium-neon source at 543 and 665 nm.