Dm6000b microscope

Hematoxylin and eosin (H&E) staining was used to evaluate the general morphology of the brain tissue and orientation of the brain regions as described previously [31 (link)]. For H&E staining, standard tissue paraffin block was sectioned at 20‐μm thickness and the slides were allowed to dry and heated at 60°C for 30 min. Before staining, sections were deparaffinized in three changes of xylene and rehydrated through graded concentrations of ethanol. Sampling areas and the strategies for identifying regions of interest were shown using standard H&E‐stained sections (Figure 1A).
Multichannel confocal triple‐labeled microphotographs were captured using a Leica SP8 spectral confocal microscope with settings appropriate for the fluorophore. Images were captured with a Leica microscope DM6000 B using a 40× objective (Leica Microsystems Ltd.) and processed in Adobe Photoshop (version 11.0.2; Adobe Systems Inc.).

The stable cell line HeLa-shPrimPol has been described before^{24 (link)}. DNA sequences encoding WT PrimPol and Y100H variant were cloned into Gateway expression vectors (Invitrogen) introducing an N-terminal V5 tag. Transient transfection was performed using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA). HeLa-shPrimPol cells growing exponentially in culture were pulse-labelled with 50 μM CldU (20 min) followed by 250 μM IdU (20 min). Labelled cells were harvested and resuspended in phosphate buffered saline at 0.25 × 10⁶ cells/mL. Stretched DNA fibers were prepared as described^{55 (link)} with minor modifications. A detailed protocol is available upon request. For immunodetection of labelled tracks, fibers were incubated with primary antibodies anti-CldU (rat monoclonal anti-BrdU, Abcam #AB6326) and anti-IdU (mouse monoclonal anti-BrdU, BD Biosciences #347580) for 1 h at RT and the corresponding Alexa Fluor-conjugated secondary antibodies (Invitrogen/Molecular Probes #A-11007 and A-21121) for 30 min, both at room temperature in a humidity chamber. DNA was stained with anti-ssDNA (Millipore, #MAB3034) to assess fiber integrity. Fiber images were obtained in a DM6000 B Leica microscope. Fork rate was estimated from > 300 red-green tracks per condition using conversion factor 1 μm = 2.59 kb^{56 (link)}.

Cells grown onto round coverslips were treated as described in the figure and incubated with 25 μM EdU for the last 30 min. The coverslips were then washed twice with PBS and cells were pre-extracted with CSK buffer (10 mM PIPES pH 7, 0.1 M NaCl, 0.3 M sucrose, 3 mM MgCl₂) for 5 min 4 °C. After fixation with formaldehyde for 10 min at room temperature, a click reaction was performed on the coverslips (100 mM Tris pH 8, 10 mM CuSO₄, 2 mM Na-l-ascorbate, 50 μM biotin-azide-AF488) for 1.5 h at 37 °C. Then, the coverslips were washed with PBS and DNA was stained with DAPI (1 mg ml⁻¹ in PBS) for 10 min at room temperature, mounted with Prolong Gold Antifade (Thermo Fisher Scientific) and visualized under a fluorescence microscope (DM6000 B Leica microscope with a HCX PL APO 40 0.75 NA objective). EdU intensity was assessed in at least 250 EdU-positive nuclei per condition per experiment using CellProfiler (v.3.1.9).

Confocal images were taken on a Zeiss LSM700 or an Olympus BX61 Fluoview microscope, whilst brightfield or Nomarski images were taken on a Zeiss AxioImager M2. Low-magnification fluorescent images of adult transgenic fish were taken on a Leica MZ16FA. In all experiments, multiple individuals or sections were examined and representative micrographs imaged.
To follow the expansion or loss of mCherry expressing cells upon permanent Cre-lox mediated labelling, floxed individuals were imaged every 4 days from 12 days onwards. Fish were anaesthetized with 0.02% Tricaine (buffered to pH 7.0) and mounted in 3% methyl cellulose. A post-anal region was iteratively re-imaged using a DM6000B Leica microscope. After imaging, the embryos were washed in facility water and returned to the fish facility.