Tcs sp5 2 laser scanning microscope

For immunofluorescence microscopy, HEK293 cells stably expressing CCR7-YFP2 grown on coverslips were transiently transfected with individual Nb-YFP1 clones. The subsequent day, cells were stimulated or not with 0.5 µg/mL CCL19, washed and fixed in 4% formaldehyde for 15 min at RT. Afterwards, cells were permeabilized using 0.2% Triton-X-100 and 0.125 % SDS in PBG (3% BSA and 20 mM Glycine in PBS) for 30 min at RT and incubated with anti-YFP1 (E385) (abcam, Cambridge, United Kingdom) and anti-YFP2 (11814460001) (Roche, Basel, Switzerland) specific primary antibodies in PBG for 1 h at RT. Cells were washed with PBS and incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen, Life Technologies) for 30 min at RT. Coverslips were mounted using Dako Fluorescence Mounting Medium (Dako, Glostrup, Denmark). Confocal images were acquired on a Leica TCS SP5 II laser scanning microscope using a 63x/1.4 NA oil-immersion objective (Leica, Wetzlar, Germany).

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).