Immunocytochemistry for Cellular Markers

For immunocytochemistry, 35 µm sections were incubated with a blocking buffer (5% normal serum/ 0.3% Triton X-100, Bio-Rad, Irvine, CA, USA) for 1 h. The tissue was serially sectioned to 35 µm thickness and immunostained with primary antibody [49 (link)]. The sections were incubated with primary anti-Iba-1 (1:200; catalog no. ab87117, abcam), anti-GFAP (1:200; catalog no. MAB360, milipore), and anti-NeuN (1:200; catalog no. 324307s, cell signaling) overnight. Nucleus staining was performed with DAPI. An Axiophot microscope (Leica, TCS SP8, Wetzlar Germany) was used for the analysis of double-stained sections. The immunodensities in the graphs were quantified using the ImageJ program software.