*2.4. Tissue Processing*

Three days after SE induction, animals were perfused transcardially with phosphate-bu ffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate bu ffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg i.p.). The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 ◦C for 2 days. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 μm, and consecutive sections were collected in six-well plates containing PBS. For western blot, animals were decapitated under urethane anesthesia. The hippocampus was rapidly removed and homogenized in lysis bu ffer. The protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit (Pierce Chemical, USA).

#### *2.5. Immunohistochemistry and Measurements of Mitochondrial Length and Neuronal Damage*

After incubation with 10% normal goa<sup>t</sup> serum (Vector, Burlingame, CA, USA), sections were incubated in a mixture of primary antibodies shown in Table 1 (in PBS containing 0.3% triton X-100) at room temperature, overnight. After washing, sections were incubated for 1 h in a fluorescein isothiocyanate (FITC, green)-, Cy3 (red)- or aminomethylcoumarin acetate (AMCA, blue)-conjugated secondary antibodies (Vector, Burlingame, CA, USA). For negative control, tissues were incubated in pre-immune serum instead of primary antibody. Negative control tissues did not show any immunoreactivity for the primary antibody (data not shown). Images were captured using an AxioImage M2 microscope or a confocal laser scanning microscope (LSM 710, Carl Zeiss Inc., Oberkocken, Germany). Individual mitochondrion length in PV cells and CA1 neurons (*n* = 20/section) was measured using ZEN lite software (Blue Edition, Carl Zeiss Inc., Oberkocken, Germany) following 3D-reconstruction. Based on our previous study [10,18], twenty-five serial images (z-stack, 1 μm) were obtained from each hippocampal section. Serial images were stacked, aligned, visualized and converted to 3D images using the ZEN lite program. Thereafter, individual mitochondrial length (long axis) was measured. In addition, Fluoro-Jade B (FJB) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed according to the manufacturer's instructions to analyze the neuronal damage. Two different investigators who were blind to the classification of tissues performed the measurement of mitochondrial length and the cell count of FJB and TUNEL positive neurons based on the lamellar structure of the hippocampus. For quantitative analysis of fluorescent intensity, sections (15 sections per each animal) were viewed through a microscope connected via Axiocam camera (Carl Zeiss Korea, Seoul, South Korea). Thereafter, fluorescent intensity measurements were represented as the number of a 256-gray scale using AxioVision Rel. 4.8 software (Carl Zeiss Korea, Seoul, South Korea). Intensity values were corrected by subtracting the average values of background noise obtained from five image inputs. The optical density was then standardized by setting the threshold levels.

## *2.6. Western Blot*

Western blot was performed by the standard protocol. The primary antibodies used in the present study are listed in Table 1. The bands were detected and quantified on ImageQuant LAS4000 system (GE Healthcare, USA). As an internal reference, rabbit anti-β-actin primary antibody (1:5000) was used. The values of each sample were normalized with the corresponding amount of β-actin. The ratio of phosphoprotein to total protein was described as the phosphorylation level.


**Table 1.** Primary antibodies used in the present study.


**Table 1.** *Cont.*

IF, Immunofluorescence; WB, Western blot.

#### *2.7. Quantification of Data and Statistical Analysis*

All data were analyzed using the Mann-Whitney test or ANOVA to determine statistical significance. Bonferroni's test was used for post hoc comparisons. A *p*-value below 0.05 was considered statistically significant.
