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2 protocols using anti p300

1

Immunofluorescent Staining Protocol

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Sample fixation was performed with a solution of 4% of paraformaldehyde in 0.1 M of PBS (Lonza) [35 (link), 36 (link)]. The following steps were performed: cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min; samples blocking with 5% skimmed milk in PBS for 30 min [37 (link)]; primary antibodies (anti-NFκB, 1 : 200, Santa Cruz Biotechnology; anti-MyD800, Thermo Fisher Scientific; anti-DNMT1, 1 : 200, EpiGentek; and anti-p300, 1 : 200, OriGene) incubation for 2 h at room temperature; and finally, secondary antibody (Alexa Fluor 568 red fluorescence-conjugated goat anti-rabbit antibody, 1 : 200, Molecular Probes, Invitrogen, Eugene, OR, USA) incubation for 1 h at 37°C. Cells were stained for 1 h with Alexa Fluor 488 phalloidin green fluorescent conjugate (1 : 400, Molecular Probes) and for 1 h with TOPRO (1 : 200, Molecular Probes) to mark the cytoskeleton actin and nuclei, respectively [38 (link), 39 (link)]. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) has been used to acquire microphotographs.
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2

Immunofluorescent Analysis of hPDLSCs

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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.
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