Laser sharp 2000

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 and blocked with fetal calf serum containing 10% (v/v) skim milk. Cells were then incubated with goat anti NQO1 C19 and R20 antibodies (Santa Cruz). Following incubation with Cy5-conjugated donkey anti-goat antibody (Jackson Immuno Reasearch Laboratories), coverslips were mounted in DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, United States ). Microscopic images were obtained using laser scanning microscope LSM710 (Carl Zeiss, Microimaging GmbH, Göttingen, Germany) with plan-apochromat 63×/1.40 oil DIC M27 objective, and managed by Laser Sharp 2000 software (Zeiss, Munich, Germany). Representative images with identical laser intensities.

The following antibodies were used for staining of the imaginal discs: mouse anti-myc 1:500 (Cell Signaling Technology, Frankfurt, Germany), guinea pig anti-H 1:250 [96 (link)], rabbit anti-mCherry 1:500 (GTX128508, GeneTex, obtained from Biozol, Eching, Germany); secondaries coupled to FITC or Cy3 were used at 1:200 (Jackson Immuno-Research, obtained from Dianova, Hamburg, Germany). Imaginal discs within the same plane were chosen, and five 1 µm thick sections taken at identical confocal settings using a Zeiss Axioskop coupled with a BioRad MRC1024 confocal microscope, using LaserSharp 2000 software (Carl Zeiss, Jena, Germany). For the quantification of signal intensity, sections were stacked, and the mean gray value of the entire disc recorded using ImageJ. Measurements of ten discs were sampled, the mean and SD (standard deviation) are indicated. Statistical analyses were performed by ANOVA for multiple comparisons, using a two-tailed Dunnett’s approach.

Primary MEFs were grown on coverslips and treated with 1 μM CPT or DMSO (control). After 1 h, the drug was removed and cells were pre-extracted for 5 min on ice in 10 mM Pipes buffer (pH 6.8) containing 300 mM sucrose, 50 mM NaCl, 3 mM EDTA, 0.5% Triton X-100, and Protease Inhibitor Mixture (EDTA-free; Roche) before fixation in 2% (wt/vol) paraformaldehyde for 15 min at 25°C. After fixation, cells were washed with PBS and then were blocked with 5% (wt/vol) BSA and 0.1% Triton X-100 in PBS before staining with mouse anti-γH2AX (Cell Signaling), rabbit anti-RPA pS4/S8 (Bethyl), and DAPI (Vector) for 1 h. After washes in PBS + 0.1% Triton X-100, Alexa 488 goat anti-mouse/rabbit, and Alexa 598 goat anti-mouse/rabbit (Molecular Probes) were used as secondary antibodies. Images were acquired using a Bio-Rad Radiance 2100 (Nikon Eclipse E800) microscope using Lasersharp 2000 software (Zeiss).