J1800amnz

Cells were treated with 20 ng ml^–1 KaryoMAX colcemid solution (Thermo Fisher Scientific, 15212012) for 2 h at 37 °C in a humidified incubator. Cells were collected in 0.8% sodium citrate solution (Sigma-Aldrich, S4641) and maintained at 37 °C for 30 min. The cell suspension was fixed with 3:1 methanol:acetic acid (Chemie Brunschwig, M/4000/17; FSHA/0406/PB08) added drop-by-drop, washed twice in the fixative solution and incubated overnight at −20 °C. Cells were dropped onto a glass slide (Thermo Fisher Scientific, J1800AMNZ). Slides were incubated for 2 min in a humidified chamber at 65 °C and air-dried at room temperature for 30 min. Slides were mounted and DAPI-stained concomitantly with ProLong Diamond antifade mountant with DAPI (Thermo Fisher Scientific,. P36962) according to the manufacturer’s instructions. Metaphases were imaged at ×100 resolution on a Zeiss Axioplan upright microscope. Images were analysed using Fiji (v.2.9.0)^{53 (link)}.

For histological analysis, 10-μm-thick cross-sections of TA or EDL muscles were obtained using a cryostat microtome (Microm HM550, Adamas Instruments, Rhenen, The Netherlands), mounted on microscope slides (super frost plus, Thermo Scientific, J1800AMNZ, Landsmeer, The Netherlands) and stored at –80 °C for further analyses. In addition, for TA muscle, 10-μm-thick longitudinal sections were obtained to measure the myonuclear length.

As detailed in previous reports [53 (link),54 (link),55 (link),56 (link),57 (link),58 (link),59 (link)], animals were anesthetized as described above and sacrificed by intra-aortical perfusion of 1 mg/kg of 4% paraformaldehyde (PFA) (P6148, Sigma-Aldrich, Steinheim, Germany). Subsequently, a 2 cm spinal cord stretch containing the lesion was extracted, post-fixed in 4% PFA for 4 h (h), cryoprotected by immersion in 30% sucrose (84100, Sigma-Aldrich, Steinheim, Germany) for 72 h, frozen embedded in Neg-50 medium (6502, Epredia, Breda, The Netherlands), and cut using a cryostat to obtain parallel transverse spinal cord sections with a thickness of 30 µm, which were mounted on slides (J1800AMNZ, Thermo Scientific, Braunscweig, Germany) and stored at −20 °C until further use.