Atto 647n

For stimulated emission depletion (STED) microscopy and the corresponding confocal microscopy a LEICA SP5-STED microscope was used. The fluorophores ATTO647N (Activemotif) or MegaRed (Sigma) were excited with an 80 MHz pulsed diode laser at 635 nm or 531 nm respectively (Pico-Quant), (pulse width < 100 ps). STED depletion was achieved using a mode-locked titanium sapphire laser (Spectra Physics Mai Tai HP laser) operating at 770 nm with a repetition rate of 80 MHz. The delay between the excitation and STED pulses was adjusted electronically to optimize depletion. The excitation and the STED beams were focused by a 100× oil immersion objective (NA 1.4 PL APO STED, 100×; Leica Microsystems). The fluorescence signal was collected by the same objective and detected confocally between 650 and 690 nm for ATTO647N and between 540 and 595 nm for Megared with an APD detector. Using this set up, a resolution of ˜250 nm in the confocal images and 50–70 nm in the STED images was achieved. Except for contrast stretching and level adjustment no further image processing was applied.

Confocal imaging was carried out using a Nikon A1R system using 60x objective (100x was used for DNA FISH images). For DNA FISH imaging, nuclei from tissue were assessed by scanning through the z axis and 2D images were acquired selecting the plane giving the strongest FISH signal. For RNA polymerase II imaging, a 2-color in-house built stimulated emission depletion (STED) microscope was used. Samples were visualized with Atto647N (Active Motif) and Oregon Green 488 (Life Technologies) fluorophore-conjugated secondary antibodies and imaged using a 100x objective. We used 635nm excitation and 750nm depletion lasers for Atto647 visualization and 485nm excitation and 592nm depletion lasers for Oregon Green 488 labels.