Mouse anti neun igg

To analyze the protein localization in the brain, 10-w-old mice were anesthetized with pentobarbital and transcardially perfused with 0.1 M phosphate buffer containing 4% paraformaldehyde. The brains were removed and fixed with the same fixative at 4°C overnight then infiltrated with 30% sucrose. Brains were embedded in the OCT compound (Sakura Finetek) and sectioned at a thickness of 20 μm using a cryostat (CM1850, Leica). For the immunostaining, the sections were reacted with rabbit anti-luciferase IgG (1:100, Promega) and mouse anti-NeuN IgG (1:500, Millipore). Then the sections were incubated with goat anti-rabbit IgG conjugated with biotin (1:2000, Millipore) and goat anti-mouse IgG conjugated with Alexa488 (1:500, Invitrogen), respectively. Subsequently the signal of luciferase was amplified with Elite ABC standard kit (Vectastain) and TSA Plus cyanine 3 system (Perkin Elmer). Fluorescent images were acquired using confocal laser-scanning microscope (TCS-SP5, Leica).

A subset of mice that survived to study endpoint (24 h or 14 days after exposure) were injected with sodium pentobarbital (75 mg/kg, IP, Fatal Plus; Patterson Veterinary), anesthesia was confirmed by lack of a response from a strong toe pinch, and the animals were perfused with heparinized 0.9% saline in 0.1 M phosphate buffer (FD Neurotechnologies, Columbia, MD, USA) followed by a 4% paraformaldehyde solution as previously described [13 (link)]. Brains were removed, kept in 4% paraformaldehyde for 6 h, and cryoprotected in 20% sucrose. Sectioning and staining of tissue were performed by FD Neurotechnologies using previously described methods [53 (link)]. Frozen brains were coronally cut at a thickness of 30 µm, and stained with Fluoro-Jade B, to visualize dying neurons, and the ionized calcium-binding adaptor molecule 1 (Iba1; rabbit anti-Iba1 IgG 1:6000; Wako Chemicals, Richmond, VA, USA) for experiment 1, or for experiment 2, immunohistochemically processed using antibodies against the neuronal nuclear protein (NeuN; mouse anti-NeuN IgG 1:600; Millipore, Billerica, MA, USA) and Iba1. For Iba1-stained tissue, cresyl violet was used as a counterstain for the visualization of anatomic landmarks.