Sp5 confocal system

The MRI of a boxer dog having spontaneously developed a brain tumor was performed at the Veterinary School of Maisont-Alfort (94-France) by Dr. P. Devauchelle and tumor samples were obtained with the consent of the dog owner. For immunofluorescence staining, cells were processed as previously described (20 (link), 30 (link)). Microscopy was performed at room temperature with the Leica SP5 confocal system, attached to a DMI6000 stand, with a 636/1.4 oil immersion objective. Images were processed with the freely available ImageJ software (http://rsb.info.nih.gov.gate1.inist.fr/ij/) and the Leica SP5 confocal system.

Cells seeded on coverslips (Thermo Fisher Scientific) were washed twice with PBS. Then, cells were fixed with 4% PFA for 30 min, permeabilized with 0.4% Triton X-100 in PBS for 30 min and blocked with 10% donkey serum in PBS (Jackson Immuno Research) for 1 h at room temperature. After blocking, cells were incubated with primary antibodies at 4 °C overnight. Subsequently, cells were washed three times with PBS, incubated with secondary antibodies at room temperature for 1 h and washed three times with PBS. Nuclei were stained with Hoechst 33342 (H3570, Thermo Fisher Scientific). Imaging was performed with a Leica SP5 confocal system. Statistical analysis of number, intensity and area of fluorescence signals were quantified by Image J software (NIH). Cells were collected from three biological replicates. Statistical significances were assessed by a two-tailed unpaired Student’s t-test. The 3D reconstruction of H3K9me3 staining as shown in Fig. 2f was performed by serial z-stack sectioning by 50-nm intervals at a conventional mode for up to 50 images with a Leica SP5 confocal system and further processed for 3D reconstruction by Imaris software (version 7.4.2) as previously described.^{90 (link)}