2.10. Cryostat sectioning

The NDG were embedded and frozen in embedding medium (Tissue‐Tek OCT compound; ProSciTech, Thuringowa Central, QLD, Australia) as described previously (Ngo et al., 2020). Four series of sections (16 μm thickness) were cut on a cryostat (HM525, Microm; Thermo Fisher Scientific, Walldorf, Germany) at −20°C, thaw‐mounted on gelatin‐coated slides and stores at −20°C until processing.

Brainstems were frozen at −20°C, and six series of coronal sections (40 μm thickness) were cut on a cryostat from the emergence of the pyramidal decussation caudally (bregma −15.48 mm) to the emergence of facial nerve rostrally (bregma −10.20 mm). Sections were placed directly into cryoprotectant (0.88 M sucrose, 0.25 mM polyvinyl‐pyrrolidone, 4.35 M ethylene glycol, 50 mM sodium phosphate buffer, pH 7.2) in a 24‐well plate and stored at −20°C until processing.