The cells used were MRC5 SV40 transformed human fibroblasts (a gift from A. Lehmann, University of Sussex), grown at 37 °C with 5% CO2 in MEM with 10% fetal calf serum (Gibco) and L-glutamine and penicillin/streptomycin (PAA). For indirect immunofluorescence cells were grown on 170 μm thick glass coverslips until 50% visual confluence. Cells were washed in CSK (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2) then the soluble proteins were removed by incubation in CSK with 0.5% triton X-100 for 3 minutes followed by a wash in CSK and fixation in 2% freshly dissolved formaldehyde in PBS. Non-specific antibody binding sites were blocked by incubation in 5% BSA in PBS with 0.1% tween20 (blocking buffer) for 30 minutes, primary antibody incubation was for 1 hour at room temperature with antibodies (anti-RPA34 monoclonal Abcam ab16855, anti-PCNA polyclonal Abcam ab18197) diluted 1/500 in blocking buffer. After three washes secondary antibodies coupled either to Alexa Fluor 488 (Molecular Probes) or Atto 647N (Sigma) (all at 1/500 dilution) were added for 1 hour in blocking buffer. After 5 washes of 5 minutes each in PBS with 0.1% tween20 the coverslips were mounted in aquapolymount.
In some cases cells were pulse labelled for ten minutes with 40 μ
M EdU (5-ethynyl-2-deoxyuridine) (Invitrogen), and after triton extraction and fixation as described above, sites of EdU incorporation into DNA were visualised by copper catalysed-click chemistry [45
] with an amide derivative of Alexa Fluor 488 according to the manufacturer's protocol. Subsequent immunofluorescence was as above.
Where used hydroxyurea treatment was for 12 hours at 2 mM followed by immediate extraction and fixation as above.
Throughout this study we selected cells that showed the characteristic staining found in early S-phase, with many evenly sized nuclear replication factory distributed evenly throughout the nucleoplasm. All images were acquired on a Leica TCS STED equipped with an inverted microscope (DMI 6000, Leica) and a 100 × STED objective (HCX PL APO 100 × 1.4 oil STED, Leica). Confocal images were acquired using the 488 nm line for the excitation of Alexa 488 and a pulsed 635 nm laser diode (PicoQuant) to excite Atto 647N. Alexa 488 was detected using PMT 2 of the spectral detection unit with the detection range set to 495 - 550 nm and Atto 647N was detected on APD 2 equipped with a 685/40 (Semrock Bright Line, Semrock) nm bandpass filter. Imaging speed was at 400 Hz using 7 × line averaging and the pinhole was set to 0.5 Airy units. At zoom 11 and a format of 512 × 512 the resulting pixel size was 27.6 nm. Z-stacks were run at a step-size of 80 nm using the galvanometric-driven fine focussing stage of the system.
For STED microscopy all conditions were identical, but additionally the depletion laser was activated. For the stimulated emission depletion of Atto 647N the pulsed Ti:Sa IR laser (Mai Tai HP, Spectra Physics) was tuned to 750 nm and the AOM set to 100%. We calculated the xy resolution limit of our STED system to be 75 nm by imaging 20 nm crimson beads and measuring the full width half maximum of the obtained images using Leica's LAS AF quantification tool.
Confocal and STED image stacks were deconvolved using SVI's Huygens Professional package. We used the CMLE (Classic Maximum Likelihood Estimation) method of this software. The CMLE method was applied using SVI's "Confocal" optical parameters set, whereas the sampling intervals were set manually to the actual experimental values (27.6 nm for X and Y pixel sizes, 80 nm for Z step size), together with the refractive indices (1.51 for oil immersion objective and medium) and excitation/emission wavelengths. The complete parameter set was saved as a template and applied for each data set. We also performed deconvolution using a calculated PSF for the STED system (provided by Leica Microsystems). The data analysed in this way did not vary significantly from the deconvolution presented here.
Object counting was performed on the 3-D image stacks using Huygens Object Analyzer. Each data set was analysed with garbage volumes set at 1, 3, 5, 10 and 50. For each garbage volume, the fluorescence threshold was set to the value that resulted in the maximum number of objects identified. The seed value was always set at 0%, thus not setting a ceiling on voxel intensity values. Objects were counted within a pancake-shaped region of interest (ROI), where the top and bottom planes of the ROI were set to the highest and lowest Z positions of non-zero voxels of all identified objects, whereas the XY outline of the ROI was drawn manually in order to exclude stray objects from the analysis. The number of objects was divided by the volume of the ROI before calculating data average and scatter. The objects were also characterised by the axial width, as defined in Huygens: the largest width in an axial direction perpendicular to the length axis. Axial width values were only included for objects inside the ROI. The resulting datasets were analysed in Excel (Microsoft Office 2007): average values were calculated with the AVERAGE function, whereas the data scatter was characterised with the AVEDEV function.
Colocalisation analysis of doubly stained cells was performed after background subtraction using the "intensity correlation analysis" plugin for Image J from the MacMaster Biophotonics facility (full details and download available from http://www.macbiophotonics.ca/index.htm