Killik oct

Tissues were prepared according to standard methods^{38 (link)}. Briefly, after induction of anesthesia with a solution containing 100 mg/kg of ketamine (Ketavet, Gellini) and 33 mg/kg of xylazine (Rompun, Bayer). When deeply anesthetized the animals were perfused with a solution containing 4% paraformaldehyde (PFA) and 2% picric acid (AnalytiCals, Carlo Erba 409302) in 0.1 M sodium phosphate buffer (PB) pH 7.4. After dissection brains were post-fixed for 3 h, cryoprotected in 30% sucrose (S0389 Sigma) in 0.1 M PB pH 7.4, embedded at − 80 °C in Killik/OCT (Bio-Optica 05-9801), and serially cut on a cryostat, into 40 µm-thick sections.

Tibialis anterior (TA) muscles were collected, embedded in optimal cutting temperature compound (Killik—O.C.T., Bio Optica, Milan, Italy), and snap-frozen in liquid nitrogen for 10 s. Embedded muscles were stored at −80 °C for transverse cryo-sectioning with a Leica cryostat. Cryosections (10 μm thickness) were collected on Superfrost glass slides (Thermo Fisher Scientific, Monza, Italy), and tissue slides were stained with hematoxylin and eosin (H&E).
For the H&E, cryosections were fixed with 4% paraformaldehyde (PFA, Santa Cruz Biotechnology, D.B.A. Italia S.r.l., Segrate Milan, Italy) for 15 min at room temperature (RT). After washing in 1X PBS, tissue slides were incubated in the hematoxylin solution for 15 min and rinsed for 5 min in tap water. Cryosections were then counterstained with an alcoholic solution of eosin for 30 min. Following the eosin staining, cryosections were dehydrated in increasing concentrations of alcohol, clarified with the histo-clear solution (Agar Scientific Ltd, Stansted, UK), and finally mounted on coverslips, using the resinous Eukitt mounting medium (Electron Microscopy Sciences, Hatfield Township, PA, USA).
H&E images were captured using the Zeiss Lab A1 AX10 microscope at the 20× magnification in the bright field.

To obtain frozen sections, fixed brains were cryoprotected in 30% sucrose in PBS and embedded into Killik O.C.T. (Bio Optica, Milan, Italy) and then placed at −80 °C for quick freezing. Brains were sectioned into 12 µm-thick slices using the HM525 NX cryostat (Epredia, Portsmouth, NH, USA) and finally collected onto polarized SuperFrost™ Plus Adhesion Microscope Slides (Epredia).
ISH on frozen tissue sections was performed as described in Gabellini et al. [25 (link)], with some modifications. Cryosections were thawed and washed in PBT (PBS + 0.5% Triton X-100) and incubated with either 300 ng/mL prr21a-201 or prr12b-201 antisense probes at 65 °C O/N. Slides were then rinsed at 65 °C in Hybe Wash and MAB + 0.1% Tween20 (Sigma-Aldrich) (MABT) solutions at RT. After 1 h-long equilibration at RT in the previously described blocking solution added with 20% lamb serum, slides were incubated with anti-DIG antibody (diluted 1:2500 in the blocking solution) at 4 °C O/N. Slides were stained in BM Purple Solution in the dark at RT. After the staining procedure, images were acquired using a Nikon Eclipse Ti microscope (Nikon Instruments).