*4.7. Immunohistochemistry*

Immunohistochemistry was conducted in the usual way to evaluate neuronal damage using NeuN antibody, and to examine oxidative stress using 8OHdG antibody, 4HNE antibody, and endogenous antioxidant (SOD1 and SOD2) antibodies (Table 1). In brief, the sections were reacted with 0.3% hydrogen peroxide and subsequently immersed in 5% normal horse serum at room temperature. After washing, these sections were incubated in each primary antibody solution overnight at 4 ◦C. Subsequently, the sections were exposed to corresponding secondary antibody, as shown in Table 1, for two hours at room temperature and to avidin-biotin complex (diluted 1:200, Vector) for one hour at room temperature. These sections were then visualized using 0.06% 3,3- -diaminobenzidine tetrahydrochloride (Sigma-Aldrich Co., St. Louis, MO, USA) for one minute, and the immunoreactions were checked. Finally, they were mounted onto gelatin-coated slides and were sealed with mounting medium.


**Table 1.** Primary and secondary antibodies for immunohistochemical staining.

## *4.8. Analyses of Data*

For quantitative analysis of IR-induced neuronal death, nine sections per animal were selected at the corresponding levels using the brain atlas of gerbil [46]. In short, as described previously [47], the digital images of NeuN- and FJB-stained cells were taken using a light microscope (BX53, Olympus, Tokyo, Japan) and an epifluorescent microscope (Carl Zeiss, Göttingen, Germany), respectively. The cell count was evaluated using Optimas 6.5 (an image analyzing system; CyberMetrics, Scottsdale, AZ, USA).

To quantitatively analyze the changes in 8OHdG, 4HNE, SOD1, and SOD2 immunoreactivities, a relative optical density (ROD) was applied. As described previously [48], in short, the digital image of immunostained structure was obtained using BX53 light microscope. The obtained image was converted into grey scale image (from black to white). The grey image was calculated by using Image J software (version 1.46) of the National Institutes of Health (Bethesda, Maryland, MD, USA).

#### *4.9. Statistical Analyses*

Statistical analyses were performed with aid of GraphPad Prism (version 5.0) (Graph-Pad Software, La Jolla, CA, USA). The differences of the means among the groups were statistically analyzed by two-way analysis of variance (ANOVA) tests with post hoc Bonferroni's multiple comparison tests to elucidate IR-related differences among the groups. Data were presented as the means ± standard errors of the mean (SEM), and statistical significance was designated when *p* value was less than 0.05.

**Author Contributions:** Conceptualization, M.-H.W. and S.Y.C.; Methodology, D.W.K. and J.H.A.; Software, M.C.S. and J.-C.L.; Validation, T.-K.L. and C.-H.L.; Investigation, J.H.P. and T.-K.L.; Data Curation, J.H.P. and J.-D.K.; Writing—Original Draft Preparation, J.H.P. and T.-K.L.; Writing—Review & Editing, M.-H.W.; Supervision, J.H.C. and M.-H.W.; Project Administration, S.Y.C.; Funding Acquisition, J.H.P., J.-D.K. and S.Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A6A1A11036849, NRF-2020R1I1A3060735 and NRF-2020R1F1A1062633).

**Institutional Review Board Statement:** The protocol of all experiments was approved (approval no., KW-2000113-1) on 13 January 2020 by the Ethics Committee of Kangwon National University (Chuncheon, Gangwon, Korea).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to appreciate Hyun Sook Kim and Seung Uk Lee for their technical help in this study.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
