Axioplan 2 fluorescence microscope

For immunohistochemistry, excised corneal samples were embedded in frozen section compounds (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 10 µm sections were cut using a HM525 NX cryostat (Thermo Scientific) and collected on polylysin-coated glass slides (Thermo Scientific). Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature. Subsequently, samples were incubated with the primary antibodies at room temperature for 1 hour or at 4 °C overnight. The primary antibodies used were Na⁺/K⁺-ATPase and ZO-1, as well as anti-human nuclei antibodies (Merck Millipore, Massachusetts, USA). Subsequently, samples were labeled with an AlexaFluor 488 conjugated goat anti-mouse IgG secondary antibody (2.5 µg/ml, Life Technology), mounted in Vectashield containing DAPI (Vector Laboratories, California, USA), and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Cells were fixed in 3.7% formaldehyde (Sigma-Aldrich) in PBS, pH 7.2 at 37°C, washed in PBS and stained against TP53 (FL-393, Santa Cruz Biotechnology) or P21 (EA10, Merck), both diluted 1∶100 in PBS supplied with 2% BSA (Sigma-Aldrich) and 0.2% Triton X-100 (Merck). Bound primary antibodies were detected using Cy3 secondary antibodies (PA43002 respective PA43004, Amersham). Slides were mounted using Prolong Gold anti-fade with DAPI (Invitrogen). Cellular fluorescence was imaged using a Zeiss Axioplan 2 fluorescence microscope (Zeiss).

To correlate UNR association with the chromosomal content (DNA FISH) or with the poly(A) tail of mRNA (RNA FISH), sequential fluorescent labeling was performed using 6-μm tissue sections, prepared from formalin-fixed paraffin-embedded human Hep-3B xenografts [10 (link)]. Firstly, UNR immunodetection was performed, images were captured and coordinates of each image were recorded to ensure repositioning of the slide to the same area after FISH assay. Secondly, interphase DNA or RNA FISH detection was performed using the same slide; the images were captured using the repositioning function of the microscope (Zeiss Axioplan 2 fluorescence microscope, Zeiss, Jena, Germany).
DNA FISH targeted the short arm of chromosome 6 (chromosome region 6p25) (SpectrumRed probe, Abbott France, Rungis, France) and the long arm of chromosome 11 (chromosome region 11q13.3) (SpectrumGreen probe, Abbott France) and was performed as previously described [13 (link)]. RNA FISH targeting polyadenylated mRNA was also performed as described [6 (link)]. At each step, slides were mounted with Vectashield antifade medium containing 4’,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France).

NIH/3T3 cells were plated at 2.5 × 10⁵ cells per well in 6-well plates with coverslips and cultured for 24 h. The cells were fixed with 3.7% formaldehyde for 10 min at room temperature. After washing with PBS, the cells were treated with ice-cold methanol for 10 min at −30 °C. After blocking with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) and washing with PBS containing 0.05% Tween^® 20, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, Alexa Fluor^® 488- or 594-conjugated secondary antibodies (Thermo Fisher Scientific) were used. Nuclei were stained using 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan). The fluorescence was observed by a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and a Leica TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany).

Healed wounds were harvested, bisected, and fixed in 4% paraformaldehyde for 12 hours at 4°C. Fixed tissue was dehydrated and embedded in paraffin blocks. 8-μm-thick sections were serially cut and incubated with a polyclonal rabbit anti-mouse anti-CD31 primary antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, followed by Alexa Fluor 594 Goat Anti-Rabbit IgG secondary at room temperature (1:200, Invitrogen) for one hour. All samples were counterstained with DAPI. Slides were mounted with the Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) and cover-slipped. A Zeiss Axioplan 2 fluorescence microscope was used to image the slides (Carl Zeiss, Inc., Thornwood, NY). Quantification of fluorescence was performed by a blinded observer analyzing at least five high-powered fields per wound at 200x using ImageJ software (NIH) as previously described [17 (link)].

Both anti-53BP1 and anti-γH2AX originated from rabbits. To correlate 53BP1 association with γH2AX, sequential fluorescent labeling was performed using 3-μm tissue sections, prepared from formalin-fixed paraffin-embedded human CdtB-Hep-3B xenografts. Firstly, 53BP1 immunodetection was performed, the stained tissue section was scanned, and the coordinates of the scanned area were recorded to ensure repositioning of the slide after γH2AX labeling. Secondly, γH2AX immunodetection was performed using the same slide; the tissue section was scanned again, using the repositioning function of the microscope (Zeiss Axioplan 2 fluorescence microscope, Zeiss, Jena, Germany). As a result (S6 Fig), CdtB activated DNA damage signaling, detected as 53BP1/γH2AX-positive foci, showing that CdtB intoxication results in γH2AX phosphorylation and 53BP1 recruitment, as previously reviewed in [35 (link)].