Orca flash 4.0 scmos camera

Images of HeLa GFP-TUBA1A cells were taken with a Nikon Ti-E inverted confocal spinning disk microscope (CSU-X1 spinning disk scan head), a × 60 oil immersion objective (NA 1.4) and a sCMOS ORCA Flash 4.0 camera (Hamamatsu). Images of microtubules were acquired with a 491 nm laser and a 500–550 nm band-pass emission filter every 5 s, using the MetaMorph software. The quantification of the maximal length of microtubules at each time point and within each cell was performed applying and an automatic threshold over the microtubule area and using the Skeletonize plugin of the ImageJ software.

Cells were washed with PBS (Sigma) and fixed in 4% paraformaldehyde for 30 min at room temperature. Then, cells were permeabilized in CSK buffer (10 mM HEPES pH 7.4, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl₂) with 0.1% Triton-X for 30 min. Blocking was performed for 1 h with 5% FBS in PBS after which cells were stained in 1% FBS in PBS overnight at 4 °C. After primary staining, cells were washed three times 5 min with PBS and incubated with secondary antibodies for 1 h at RT. After another three 5 min washes in PBS, slides were mounted with Aqua-Poly/Mount (Polysciences). Images were obtained using an AxioObserver Z1 confocal spinning-disk microscope (Zeiss) equipped with an AxioCam HRm CCD camera (Zeiss) or a sCMOS ORCA Flash 4.0 camera (Hamamatsu) and a Plan/Apo 63 Å~/1.4 water‐immersion objective. The percentage of cells with nucleolar retention of γH2AX foci were defined as cells that show an overlap (yellow) of the γH2AX (green) with the NPM (red) signal. Blinded visual scoring of rDNA damage retention was performed by two individuals. Graphs were generated using Graphpad Prism and Inkscape.

Tissues were collected into formalin, fixed for 24 h and then moved to 70% EtOH before casting into paraffin blocks. Sections were cut onto silane-coated glasses and deparaffinated. Alexa Fluor 647-labeled DNA probe reacting with a region of 16S-RNA gene common to all eubacteria (EUB-338: 5′-GCTGCCTCCCGTAGGAGT-3′) was applied onto the sections in hybridization buffer (20 mM Tris-HCl, 0.9 M NaCl, 0.1% SDS, 20% formamid, pH 7.4). Alexa Fluor 555-labeled scramble probe (5′-AGCCGTGTTGCCGTAGCC-3′) was used as a control (Supplementary Fig. 3). Sections were topped with a cover glass and incubated at 50 °C o/n. Cover glass was then removed and sections were washed once with wash buffer (20 mM Tris-HCl) and 3 times with PBS. Stained sections were mounted with ProLong Diamond Antifade Mountaint with DAPI (Molecular Probes, USA) and visualized with Nikon Eclipse Ti-2 microscope (Japan) and photographed with Hamamatsu sCMOS Orca-Flash4.0 camera (Japan).

Immunofluorescence staining was performed as described previously^{47 (link)}. In brief, cells were fixed with 2% paraformaldehyde + 0.1% Triton X-100 for 15 min at room temperature, washed 3× with PBS + 0.1% Triton X-100 and subsequently permeabilized with PBS + 0.1% Triton X-100 for 2×10 min at room temperature. After blocking the cells in PBS + (PBS + 0.5% BSA + 0.15% glycine), cells were incubated with the primary antibody (see Supplementary Table 5), diluted in PBS + , overnight at 4 °C. Unspecific antibody staining was removed by washing the cells 5× in PBS + 0.1% Triton X-100. Subsequently, cells were stained with Alexa Fluor 488/568-conjugated fluorescent secondary antibody (Thermo Fisher), diluted 1:500 in PBS+, for 1 h at room temperature. Finally, cells were washed 5× with PBS + 0.1% Triton X-100, stained with Hoechst 33342 (Fisher Scientific, 1:5000 in PBS) for 10 min, and washed 3× in PBS. The immunofluorescence intensities were measured on a Zeiss Axio Observer Z1 confocal spinning-disk microscope equipped with an sCMOS ORCA Flash 4.0 camera (Hamamatsu), using a Plan-Apochromat ×40/0.95-KOrr air objective or a ×40 C-Apochromat/1.2-KOrr water objective.

Mouse embryos at gestational stage E12.5 (stage of early lung branching) were removed from homozygous Muc5b-GFP mothers. Embryos were fixed and embedded in paraffin for further immunohistochemical analyses. Whole embryonic lungs were also dissected from the embryos and embryonic whole lung cultures were performed as described elsewhere13 (link). Recombinant IL13 (10 ng/mL; SRP4166, Sigma Aldrich, France) was added into the culture medium on day (D) 0. Lung explants were cultured at 37 °C in 5% CO₂ for up to 11 days. Two sets of embryos were used and pooled for the statistical analysis. Photographs were taken in bright field (50 ms acquisition) and fluorescent mode (10 sec acquisition without binning at maximum light brightness) using a Leica M205 stereomicroscope equipped with a sCMOS ORCA-Flash4.0 camera (Hamamatsu) at x30.3 magnification without a change in depth or focus. The lung area was measured using ImageJ/FIJI14 (link) and normalized to the area at D0. Fluorescence activity was quantify using FIJI and expressed as intensity/area and normalized to the fluorescence activity measured at D0.