Nuclear green dcs1

The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with methanol at −20 °C for 10 min. After blocking with 1% bovine serum albumin, the primary antibody against p65 (8242S; Cell Signaling Technology) was applied at a 1:400 dilution for 1 h. Then, the secondary antibody conjugated with Alexa Fluor 568 (Abcam, Cambridge, UK) was applied at a 1:1000 dilution with 3 μM Nuclear Green DCS1 (Abcam) for 30 min. After mounting on glass slides, the subcellular localization of p65 and the nucleus was visualized with FL2 (orange-red) and FL1 (green) detectors, respectively, using Nikon BioStation IM (NIKON, Tokyo, Japan) [14 (link),26 (link)]. The brightness of green and orange-red fluorescence was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The amount of p65 in the nucleus was calculated as the ratio of the brightness of orange-red fluorescence to that of the corresponding green fluorescence.

The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with methanol at -20 °C for 10 min. After blocking with 1% bovine serum albumin, primary antibody against p65 (8242S; Cell Signaling Technology, Danvers, MA, USA) was applied at a 1:400 dilution for 1 h. Secondary antibody conjugated with Alexa Fluor 568 (Abcam) was applied at a 1:1,000 dilution with 3 μM Nuclear Green DCS1 (Abcam) for 30 min. After mounting, subcellular localization of p65 and nucleus was visualized using a Nikon BioStation IM (NIKON, Tokyo, Japan) with FL2 (orange-red) and FL1 (green) detectors, respectively [15 ].

Cells were seeded directly in presence or absence of IFN-γ/TNF-α on glass slices in 6-well plates to a confluency of approximately 50%. Twenty-four hour later, cells were washed in PBS and fixed for 10 min in 4% formaldehyde /PBS. After two washing steps in PB buffer (PBS, 3% BSA), cells were permeabilized for five minutes in PBGT buffer (PBS, 0.2% Triton X-100, 20 mM Glycine and 3% BSA). After two subsequent washing steps with PBG buffer (PBS, 20 mM Glycine, 3% BSA), cells were incubated for 2 hours in the dark with the following primary antibodies, diluted in PBG: anti-SUMO-1-21C7 mouse monoclonal antibody^{55 (link)} (1:50), anti-PML (Abcam, ab179466, rabbit mAb, 1:500) and Phalloidin Alexa Fluor® 647 (Invitrogen, A22287, 1:250). Nuclear staining was performed with Nuclear Green DCS1 (Abcam, ab138905, 1:2000). Slices were carefully washed four times with PB buffer and then incubated for one hour in the dark with the secondary antibodies anti-mouse-Alexa 488 (Life Technologies, 1:250) or anti-rabbit-Alexa 568 (Life Technologies, 1:250). After four wash steps with PBS, slices were imbedded with fluorescent mounting medium (Dako, E3023) and imaged on a Leica TCS SP5 II laser scanning microscope using a ×63/1.4 NA oil-immersion objective (Leica).