For the analysis of neurite outgrowth, developing neurons at 9 DIV were replated into sterile cloning cylinders (O.D. 8 × H 8 mm2; Merck, Cat. No. CLS31668), located in the middle of Matrigel-coated glass coverslips (22 mm diameter, Cat. No. 631.0159, WDR), into six-well plates. Neurons were seeded at a density of 2500 cells/cylinder and were kept in culture accordingly to the differentiation protocol. The day after seeding, the cylinder was gently removed, allowing the radial extension of neurites by developing neurons. At 20 DIV, neurons were fixed with 3.7% paraformaldehyde in PBS for 10 min, permeabilized with ice cold 0.1% Triton X-100 in PBS and incubated in blocking buffer (5% Normal Donkey Serum; Abcam, Cat. N. ab7475) for 30 min at room temperature. Nuclear bodies and neurites were stained overnight at +4 °C with an anti-tubulin III antibody (1:1000; Abcam, Cat. No. ab18207), followed by incubation with Donkey Anti-Rabbit IgG Alexa Fluor 488 (1:1000, Abcam, Cat. No. ab150073). Images were acquired using ZEISS Axio Zoom.V16 Microscope and processed using Zeiss ZEN 2.6 Blue Microscopy Software.
To quantify axonal growth, we adapted the Sholl method of concentric rings to our cultures. Prior to processing, images were modified with ImageJ v1.5.3 (National Institutes of Health, Bethesda, MD) to eliminate fluorescence background and artefacts. For application of the Sholl analysis, we employed the ShollAnalysis ImageJ plug-in, which counts the number of intersections of neurites as a function of distance from the cell soma or explant. Immunofluorescent images were imported into ImageJ and converted in greyscale, the size scale was set accordingly, and the brightness/contrast threshold was selected manually to remove the thinner and shorter neurites emerging from the clusters. Sholl analysis was performed by selecting the centre of the neuronal body cluster as the centre of outgrowth (start radius = 0 inch) and using a step size of 0.5 inch and an end radius of 8 inch. These parameters were chosen to divide the image into concentric annuli at a radial distance of 1000 μm from each other. Intersections were counted from the border of the neuronal body cluster (set as 0 μm) outwards. Results were inputted into GraphPad Prism 9.2.0 software and statistical analysis was performed between CT and FRDA groups at each individual distance from the body cluster (two-way ANOVA with Bonferroni’s test for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001).
To quantify axonal growth, we adapted the Sholl method of concentric rings to our cultures. Prior to processing, images were modified with ImageJ v1.5.3 (National Institutes of Health, Bethesda, MD) to eliminate fluorescence background and artefacts. For application of the Sholl analysis, we employed the ShollAnalysis ImageJ plug-in, which counts the number of intersections of neurites as a function of distance from the cell soma or explant. Immunofluorescent images were imported into ImageJ and converted in greyscale, the size scale was set accordingly, and the brightness/contrast threshold was selected manually to remove the thinner and shorter neurites emerging from the clusters. Sholl analysis was performed by selecting the centre of the neuronal body cluster as the centre of outgrowth (start radius = 0 inch) and using a step size of 0.5 inch and an end radius of 8 inch. These parameters were chosen to divide the image into concentric annuli at a radial distance of 1000 μm from each other. Intersections were counted from the border of the neuronal body cluster (set as 0 μm) outwards. Results were inputted into GraphPad Prism 9.2.0 software and statistical analysis was performed between CT and FRDA groups at each individual distance from the body cluster (two-way ANOVA with Bonferroni’s test for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001).
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