Axiovision 4

For sister chromatid exchange and breakage analyses, methanol-acetic acid fixed preparations were imaged using a Zeiss Axioplan 2 microscope with a 100× objective lens. Images were analysed using AxioVision 4.7 (Zeiss). For FANCD2 images taken by DeltaVision, 36 sections (0.2 μm per section) images were taken. Images were deconvolved, and projected in 2D using SoftWoRx 4.1. Percentage of cells with symmetrical FANCD2 spots were scored and plotted. Obvious symmetrical FANCD2 spots intensity were further measured using the polygon function of SoftWoRx 4.1. For BLM fibers scoring and intensity measurements, images were captured using Zeiss Axio Imager M1 microscope and processed by AxioVision 4.7 (Zeiss). Percentage of cells with BLM fibers were scored and plotted. Line profiles across the fibers in the cells were analysed using ImageJ as described before [44 (link)].

Adult flies were aged to between 1 and 2 days old for all wing measurement experiments. A single wing was cut from each fly and stored in isopropanol for 1 hour before being mounted in DPX (Sigma cat. no. 06522). All results shown are for male wings, however, female wings also showed the same phenotypes. Mounted wings were measured using Axiovision 4.7 on an Axioplan microscope (Carl Zeiss). Imaginal discs were dissected in PBS from 120hr old L3 larvae, aged using a 1 hour egg lay. Dissected discs were mounted in 85% Glycerol on Poly-L-Lysine slides. Imaginal disc area was measured using Axiovision 4.7 on an Axioplan microscope (Carl Zeiss). Salivary glands were prepared and imaged using the same methodology as used for imaginal discs.

The muscle fiber cross-section area (CSA) was assessed for each histological section
under a light microscope (AxioVision 4.7, Carl Zeiss, Germany), using morphometric
analysis software (AxioVision 4.7.1.0, Carl Zeiss). The fiber CSA for each muscle was
obtained from digital images (40X) by measuring the area of 100 fibers located in the
central region of the section. A blind procedure was used for measurements.

Cells were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde for 1 h at room temperature, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 1 h at room temperature. Neurite outgrowth was analyzed by measuring the total length of neurites in an Axiovert microscope with the AxioVision 4.6 imaging system (Carl Zeiss, Oberkochen, Germany).
To determine cell death, neurons were maintained overnight in serum-free medium and then treated with 50 µg/mL L1 antibody 557 and exposed to oxidative stress by the addition of 10 µM H₂O₂ for 24 h. Live and dead cells were then stained with calcein-AM (ThermoFisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of three wells per condition and experiment using ImageJ (version 1.53q; https://imagej.nih.gov/ij/index.html; RRID:SCR_003070, 30 March 2022).

To determine neurite outgrowth, cells were treated with cell-penetrating tat-LIR_WT or tat-LIR_mut peptides (50 µg/mL) and 557 antibody (50 µg/mL) 30 min after seeding. After 24 h in culture, cells were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde for 30 min at room temperature, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 30 min at room temperature. Neurite outgrowth was analyzed by measuring the total length of neurites with a Zeiss AxioObserver.A1 microscope with the AxioVision 4.6 imaging system (Carl Zeiss, Oberkochen, Germany). For neurite outgrowth analysis for each condition, at least 100 neurons were counted.
To determine cell death, neurons were maintained overnight in serum-free medium and then treated with cell-penetrating tat-LIR_WT or tat-LIR_mut peptides (50 µg/mL) and L1 antibody 557 (50 µg/mL) and exposed to oxidative stress by the addition of 10 µM H₂O₂ for 24 h. Live and dead cells were then stained with calcein-AM (Thermo Fisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of the three wells per condition and experiment using ImageJ.