Axiom1 light microscope

CV histochemical staining was performed to investigate cellular distribution and morphology. In brief, according to the authors' published method (31 (link)), CV acetate (Sigma-Aldrich; Merck KGaA) was dissolved (1%) in distilled water (DW) and glacial acetic acid was added to this solution. Sections of each group were stained with CV solution and dehydrated with serial ethanol. The sections were examined using an AxioM1 light microscope (Carl Zeiss AG).

To obtain the accurate data for immunoreactivity, the sections from sham- and ischemia-operated young and adult animals were used at designated time points (sham, 1 day, 4 days and 7 days after reperfusion) under the same conditions. According to the above-mentioned western blot analysis method of p63, immunohistochemistry for rabbit anti-p63 (1:200; Abcam, Cambridge, UK) was performed under the same incubation temperature and time. In order to establish the specificity of the immunostaining, a negative control test was carried out with only the secondary antibody without primary antibody. The negative control resulted in the absence of immunoreactivity in any structures.
Eight sections per animal were selected to quantitatively analyze p63 immunoreactivity. Digital images of the hippocampus were captured with an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. According to the method of our previous study (Lee et al., 2014), semi-quantification of the immunostaining intensities was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). The level of immunoreactivity was scaled as –, ±, + or ++ representing no staining (gray scale value C200), weakly positive (gray scale value 150–199), moderate (gray scale value 100–149), or strong (gray scale value B99), respectively.