Dfc390 camera

S9.6 (hybridoma cell line HB-8730) and RNaseH1 (15606-1-AP; Proteintech) immunofluorescence (IF) was performed 48 h after siRNA transfection. Cells were fixed with ice-cold methanol, blocked and subsequently incubated with the primary and secondary antibodies. Nuclei were stained with DAPI. The S9.6 signal in nucleoli was subtracted from the integrated nuclear S9.6 signal to perform the analysis. Immunofluorescence images were acquired using a Leica DM6000 wide-field microscope equipped with a DFC390 camera at x63 magnification using the LAS AF software (Leica). Microscopy data analysis was performed using the Metamorph v7.5.1.0 software (Molecular Probes).

For visualisation of DNA–RNA hybrids, S9.6 (hybridoma cell line HB-8730) and nucleolin immunofluorescence was performed^{80 (link)}. Cells were treated with a pre-extraction solution (0.5% Triton X-100, 20 mM HEPES-KOH, 50 mM NaCl, 3 mM MgCl_2, and 300 mM sucrose) before methanol fixation. Subsequently, coverslips were incubated with anti-DNA–RNA hybrid (S9.6) and anti-nucleolin antibody (Abcam, ab50279; both 1:1000 in blocking solution) diluted in blocking buffer (2% BSA in PBS) overnight at 4 °C. After three washes with 1× PBS coverslips were incubated with secondary antibodies (anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594, 1:1000, Invitrogen, A11008 and A11005, respectively) for 1 h at room temperature. After washing as before, nuclei were counterstained with DAPI, coverslips were dried and mounted. Images were acquired using a Leica DM6000 microscope equipped with a DFC390 camera (Leica) at ×63 magnification, all the fields imaged were randomly chosen in the DAPI channel. Automatised quantitation was performed using the Metamorph v7.5.1.0 software (Molecular Probes), including the final analysis where the S9.6 signal in the nucleoli was subtracted from the integrated nuclear S9.6 signal.

DNA combing was performed as described^{65 (link)}. Briefly, cells were pulse-labeled with 25 μM iododeoxyuridine (IdU) for 20 min and with 200 μM chlorodeoxyuridine (CldU) for 20 min. Anti-ssDNA from Developmental Studies Hybridoma Bank was used instead of the one described. Fork Symmetry was calculated as IdU/CldU ratio. Fork Velocity was calculated as reported^{66 (link)}. Images were acquired with a Leica DM6000 microscope equipped with a DFC390 camera (Leica) at 40× magnification. Metamorph software (version v7.5.1.0) (Molecular Probes) was used for region measurements.