Mature neurons and microglia were visualized by immunohistochemical staining for NeuN and Iba-1 as described previously [61 (link)]. In brief, animals were re-anesthetized with isoflurane (Baxter, Deerfield, IL, USA) 4 d after ischemia, and transcardiac perfusion was performed with physiological saline and 4% paraformaldehyde. Thereafter, the brain was cryo-freezed and coronally sectioned (30 μm thick) based on gerbil atlas between 2.0 and 2.7 mm caudal to the bregma [62 (link)]. The antibodies used were mouse anti-NeuN antibody (1:1000; Merck Millipore, Temecula, CA, USA), rabbit anti-Iba-1 antibody (1:500; Fujifilm Wako Pure Chemical Corp., Osaka, Japan), goat anti-rabbit IgG (1:200, Vector, Burlingame, CA, USA), and goat anti-mouse IgG (1:200, Vector). Finally, immunoreactive signals were visualized by reaction with 3,3-diaminobenzidine tetrachloride (Sigma-Aldrich).
NeuN-immunoreactive nuclei were counted in the hippocampal CA1 region using OPTIMAS software (version 6.5; CyberMetrics® Corporation, Phoenix, AZ, USA). Iba-1 immunoreactivity was assessed based on the intensity and pixel number of immunoreactive signals using ImageJ software (version 1.80; National Institutes of Health, Bethesda, MD, USA) as described previously [61 (link)].
NeuN-immunoreactive nuclei were counted in the hippocampal CA1 region using OPTIMAS software (version 6.5; CyberMetrics® Corporation, Phoenix, AZ, USA). Iba-1 immunoreactivity was assessed based on the intensity and pixel number of immunoreactive signals using ImageJ software (version 1.80; National Institutes of Health, Bethesda, MD, USA) as described previously [61 (link)].
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