He cy3

Samples for microscopic observation were prepared the same way as previously described (49 (link)). Images were acquired and processed using a Zeiss Axioplan 2 imaging system with a Zeiss Imager M2 and an AxioCam 506 camera. GFP was visualized using filter set 38, HE GFP (Carl Zeiss Microscopy). The mCherry signal was visualized using filter set 43, HE Cy 3 (Carl Zeiss Microscopy).

Cells seeded, grown, and treated on glass coverslips were fixed in 4% paraformaldehyde in 0.2 M HEPES for 1 h at room temperature and permeabilized with blocking solution (PBS supplemented with 0.1% saponin, 0.5% BSA and 50 mM NH₄Cl) for 30 min at room temperature. Cells were incubated with primary antibodies, specific Alexa 488- and 546-conjugated secondary antibodies and Hoechst 33542 diluted in blocking solution. For imaging, samples were examined using a Zeiss (Oberkochen, Germany) Imager A2 microscope, equipped with 49 DAPI (excitation 365, beam splitter FT 395, emission BP 445/50), 43 HE CY3 (excitation BP 550/25, beam splitter FT 570, emission BP 605/70), and 38 HE EGFP (excitation BP 470/40, beam splitter FT 495, emission BP 525/50) filter sets (Zeiss). Images were obtained under a 20x/0.50 Plan-Neofluar M27 objective (Zeiss), at a definition of 1388 × 1040 pixels (150 dpi), by means of a high-resolution monochromatic camera (Axiocam MRm Rev3, Zeiss), and analyzed with the Axiovision REL 4.7 software (Zeiss).

Cells seeded, grown, and treated on glass coverslips were fixed in 4% paraformaldehyde in 0.2 M HEPES for 1 h at room temperature and permeabilized with blocking solution (PBS supplemented with 0.1% saponin, 0.5% BSA and 50 mM NH₄Cl) for 30 min at room temperature. Cells were incubated with primary antibodies, specific Alexa 488- and 546-conjugated secondary antibodies and Hoechst 33542 diluted in blocking solution. For imaging, samples were examined using a Zeiss (Oberkochen, Germany) Imager A2 microscope, equipped with 49 DAPI (excitation 365, beam splitter FT 395, emission BP 445/50), 43 HE CY3 (excitation BP 550/25, beam splitter FT 570, emission BP 605/70), and 38 HE EGFP (excitation BP 470/40, beam splitter FT 495, emission BP 525/50) filter sets (Zeiss). Images were obtained under a 20x/0.50 Plan-Neofluar M27 objective (Zeiss), at a definition of 1388 × 1040 pixels (150 dpi), by means of a high-resolution monochromatic camera (Axiocam MRm Rev3, Zeiss), and analyzed with the Axiovision REL 4.7 software (Zeiss).