Axio imager z1 microscope x40 objective

After indicated treatments and deposition on slides as described above, cells were fixed with 4% PFA for 10 min at RT, permeabilized with 0.1% Triton in PBS-0.5% BSA for 10 min, and saturated with PBS-1% BSA for 20 min. Anti-nucleolin (ab22758, Abcam) and anti-fibrillarin (ab5821, Abcam) antibodies were diluted 1/200 in saturation buffer, and incubated for 90 min in a humid chamber at room temperature. Slides were washed 3 times with PBS-0.01% Tween-20 and anti-rabbit Alexa Texas Red-conjugated antibody (diluted 1/500 in saturation buffer) was added for 45 min in a humid chamber at room temperature protected from light. Slides were washed 3 times (5 min each) as previously, incubated with DAPI (20 µg/mL in water) for 5 min protected from light, washed 3 times with water, air dried and mounted with ProLong (Invitrogen). Images and fluorescence were captured with a ZEISS Axio Imager Z1 microscope (X40 objective) (Oberkochen, Germany), and analyzed with Omero (omero.mri.cnrs.fr) server. Quantification of cells with nucleolin delocalization was visually performed.

Cells were treated with UNC-0379 3 µM or the vehicle DMSO (0.0003%) for 24 h. EU (0.2 mM) was added during the last 5 min of treatment. Cells were deposited on slides as described above, fixed with 4% PFA for 10 min at RT, permeabilized with 0.5% Triton in PBS-0.5% BSA for 10 min, saturated with PBS-1% BSA for 20 min and Click-it reaction was performed. Briefly, for 10 slides, a mix of 438 µL PBS 1X + 15 µL CuSO₄ (0.1 M) + 2.5 µL Azide AF555 + 50 µL Vitamine C (100 mM) was prepared by adding the reagents in the mentioned order. Slides were incubated in a humid chamber protected from light for 15 min at RT, washed 3 times with PBS-0.01% Tween-20, and subsequently incubated with DAPI and mounted as described above. Image acquisition was performed with a ZEISS Axio Imager Z1 microscope (X40 objective) (Oberkochen, Germany), and analyzed with Omero (omero.mri.cnrs.fr) server. Quantification of EU intensity was performed with CellProfiler™ “Human Cell” pipeline.