High magnification optical microscope

After antigen obtainment, the 4 μm-thick slices were incubated with antibodies against Nrf2 (1:200; Cell Signaling Technology), NeuN (1:100; Boster Biotech, Wuhan, China), LC3 (1:100; Cell Signaling Technology) and HDAC3 (1:100; Cell Signaling Technology) overnight at 4°C. Following washing, they were then incubated with secondary antibodies for an additional 1 h at room temperature. The cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). A pathologist who is blind to experiments randomly selected five ROI under a high magnification optical microscope (400×; Leica) to observe the positive staining cells surrounding injury areas. Five random ROIs were selected for quantification and the mean was used for the statistical analysis.

The cortical tissue in the lesioned areas were fixed in formaldehyde and embedded in paraffin. The tissue blocks were sectioned at a thickness of 3 μm. After de-paraffinization with dimethyl benzene and dehydration in a series of graded alcoholsolutions, the antigen was obtained via the citric acid buffer/microwave method. The sections were blocked with goat serum and incubated overnight with primary antibodies against HDAC3 (1:100; Cell Signaling Technology, Danvers, MA, USA), Iba-1 (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and GFAP (1:200; Abcam, Cambridge, UK). Following washings, slices were incubated with secondary antibodies for an additional 1 h at room temperature. A pathologist who was blinded to the experiments randomly selected five regions of interest (ROI) under a high magnification optical microscope (400×; Leica, Wetzlar, Germany) to observe positively staining cells surrounding injury areas. Evaluation of sections was undertaken by assessing the intensity of staining (five grades; Zhang et al., 2016 (link)) where: 0 indicated no detectable positive cells; 1 indicated very low density of positive cells; 2 indicated a moderate density of positive cells; 3 indicated a higher, but not maximal density of positive cells; and 4 indicated the maximal density of positive cells. A mean of five ROIs were used for statistical analysis.