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.
Free full text: Click here
