Dapi fluoromount g mounting medium

Differentiated glioblastoma cells were seeded in 24-well plates (Sarstedt, Nümbrecht, Germany) with a density of 1500 cells/well, on glass coverslips (Langenbrinck GmbH, Emmendingen, Germany). Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 min. The cells were then permeabilized with 20% Methanol for 15 min. To block unspecific binding of antibody, cells were then incubated with 20% Bovine serum albumin (BSA) at RT for 60 min. Primary antibody mix (200 μL; rabbit polyclonal anti-connexin-43 (Sigma, St. Louis, MO, USA) and rabbit monoclonal ß-tubulin (Cell signaling, Cambridge, UK), dilution 1:1000) was added to each coverslip containing wells and incubated at 37 °C for 60 min. Incubation with secondary antibodies (goat anti-rabbit Alexa Fluor 488 and Alexa Fluor 647 (Sigma, St. Louis, MO, USA), dilution 1:1000) was performed at RT for 60 min. DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) was used for nuclei staining. Images were taken with the use of AX 70 microscope and processed with Zeiss Zen software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Fluorescence intensity was calculated as follows: integrated density—(Area of selected cells × mean fluorescence of background).

Stable or transfected cells were directly grown on coverslips for at least twenty-four hours prior to fixation in cold methanol for 10 min at -20°C (HeLa or HEK293T cells, respectively). When indicated, a prior permeabilization step was carried out to evidence the strong association of ISG20 to nuclear bodies. In this case, cells were transfected in duplicate and while one aliquot was immediately fixed, the other was first washed in cold wash buffer (PBS, 0.2% Tween 20) and further permeabilized in cold PSB containing 0.5% Triton-X100 for 5 min on ice. Cells were then fixed and incubated with the indicated antibodies (see relevant section above). DAPI Fluoromount G mounting medium was used (0100, Southern biotech). Images were acquired using a spectral Zeiss LSM710 or LSM800 confocal microscopes and analyzed using the Fiji software. For P bodies analyses, cells were fixed first in 4% paraformaldehyde, then in 70% methanol (10 min each), followed by permeabilization in cold PSB containing 0.5% Triton-X100 for 5 min on ice.