Everbrite hardset mounting medium

RNAScope^® technology (Advanced Cell Diagnostics Milan, Italy) was used for in situ RNA hybridization according to the manufacturer's protocol. The sections were washed in 1x PBS + 0.1% triton for 3 × 5 min and then placed on glass slides and air dried. The slides were incubated in warmed pre-treatment 3 (Advanced Cell Diagnostics Milan, Italy) for 5 min, kept at a light boil, and then placed in distilled water. The slides were then subjected to RNAscope^® Multiplex Fluorescent Assay by first incubating the probe (mmOlig2, mmCasp3, or mm MOG) for 2 h at 40°C and then pre-forming the amplification steps according to instructions. The slides used for Olig2/EdU labeling were then blocked with 5% normal goat serum in 1x PBS +0.1% triton at room temperature for 1 h, and the Click-iT^® assay (described above) was then performed followed staining with the nuclear marker DAPI for 5 min. The sections were washed and mounted (Everbrite Hardset mounting medium, Biotium, Hayward, CA).

GFP-tagged ataxin-3 constructs were transfected in SH-SY5Y neuroblasts grown on collagen-coated 18 mm glass cover slips. 24 Hours after transfection, cells were fixed with 4% paraformaldehyde for 10 minutes and washed with PBS containing 0.5% bovine serum albumin (Sigma-Aldrich) and 0.15% glycine (Thermo Fisher Scientific) for 15 minutes. Slides were then incubated with rabbit anti-53BP1 at 1:1000 (Novus Biologicals, Littleton, USA) for 1.5 hours. After washing, detection was performed by incubation with anti-rabbit IgG Alexa 546 1:1000 (Invitrogen) for 1 hour and mounted with EverBrite hardset mounting medium containing DAPI (Biotium, Hayward, USA). Images of >50 cells were obtained the next day using an AxioImager M2 (Zeiss, Oberkochen, Germany) equipped with a 63x PLAN APO (1.4 NA) oil-immersion objective and an HXP 120 metal-halide lamp used for excitation. Foci were quantified using an ImageJ custom-made macro as described previously47 (link). Only cells expressing the GFP proteins were included for analysis.

The rats were euthanized with isofluorane (3.5–5%, 3.9–5.9 L/min) at the end of the experiment, and the sciatic nerve was harvested and fixed with 10% formalin at room temperature overnight. The sample was embedded in paraffin and pasted on a glass slide (thickness = 7 μm), followed by the removal of paraffin, rehydration, and antigen retrieval (120 °C, 10 min). The sections were washed with tris buffered saline (TBS) that contained 0.025% Triton X-100 for 10 min and were blocked with 10% newborn calf serum and 1% bovine serum albumin in TBS for 2 h at room temperature. Primary antibodies were added to the sections to react overnight at 4 °C followed by the addition of fluorescence-labeled secondary antibodies. After 2 h at room temperature, the samples were washed with TBS and mounted with the EverBrite™ hardset mounting medium that contained DAPI to label the nuclei (1:5000, 40043, Biotium, Fremont, CA). The following primary antibodies were used: anti-MBP (1:1000, ab40390, Abcam, Cambridge, UK) and anti-GFAP (1:1000, Thermo Scientific, Waltham, MA, USA). The secondary antibodies used were Alexa Fluor 555-conjugated goat antimouse IgG (1:100; Thermo Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat antirabbit IgG (1:100; Thermo Scientific, Waltham, MA, USA). Slides were viewed and images were captured with an LSM780 confocal microscope (Zeiss, Jena, Germany).