Immunohistochemical Analysis of Neuronal Markers

Immunohistochemistry was conducted as routinely executed (Chen, Zhang, et al., 2015 (link); Huang et al., 2015 (link)). The rat pups were perfused under anesthesia with PBS followed by 4% formaldehyde. The brains were removed for postfixation in formalin. The paraffin-embedded brains were then sectioned into 10 µm slices via cryostat. The brain slices were then evaluated with immunohistochemistry and a Fluoro-Jade C staining kit (Biosensis). The specific cellular marker antibody used was NeuN (1:100; Abcam). The brain slices were then incubated with the appropriate fluorescent secondary antibodies (Jackson ImmunoResearch). The brain slices were then visualized with a fluorescent microscope (Olympus BX51 and Keyence BZ-9000) under 20×, 40×, and 60 × magnifications with an aperture of 0.75, 0.95, and 0.95, respectively.