Mouse anti neun antibody

Immunohistochemical staining for NeuN, GFAP, and Iba-1 was performed to visualize surviving neurons, astrocytes, and microglia in the hippocampus 4 d after ischemia. The animals were re-anesthetized with isoflurane and perfused transcardially with physiological saline and 4% paraformaldehyde via the left ventricle. The part of the brain located on the gerbil atlas 1.4–2.0 mm caudal to the bregma [42 (link)] was sectioned at a thickness of 30-μm using a sliding microtome (HM430, Thermo Scientific, Waltham, MA, USA). Five sections, at 90-μm intervals from each other, were incubated with mouse anti-NeuN antibody (1:1000; EMD Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; EMD Millipore), and rabbit anti-Iba-1 antibody (1:500; Wako, Osaka, Japan) for 48 h at 4°C. After sequential treatment with biotinylated goat anti-mouse IgG or anti-rabbit IgG and streptavidin–peroxidase complex (1:200; Vector, Burlingame, CA, USA) for 2 h at 25°C, immunoreactions were developed using 3,3-diaminobenzidine tetrachloride (Sigma).

Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and perfused transcardially with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). Brain tissue was collected and post-fixed for 6 h in 4% PFA. For cryoprotection, tissue was transferred to 30% sucrose and stored at 4°C. Coronal sections were cut at a thickness of 40 µm using a sliding microtome (Microm HM430, Leica), collected, and stored as free-floating sections in cryoprotectant solution at −20°C.
For immunostaining, brain sections were blocked with 5% normal donkey serum for 1 h at room temperature and incubated with primary antibodies (rabbit anti-GFAP 1:200, Dako, #GA524; goat anti-Iba1, 1:500 Wako, #011-27991; NeuroTrace™ 1:200, Thermo Fischer; mouse anti-NeuN Antibody 1:500, Merck, #MAB377; rabbit anti-Neurofilament 200 antibody 1:200, Merck, #N4142; guinea pig anti-Neurofilament L, 1:200, Synaptic Systems, rat anti-CD31 antibody 1:50, BD Biosciences, #MEC13.3; goat anti-CD13, 1:200; R&D Systems, #AF2335) overnight at 4°C. The next day, sections were incubated with corresponding secondary antibodies (1:500, Thermo Fischer Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Mounting was performed using Mowiol.

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)].