Immunohistochemical Analysis of Microglia

Immunohistochemistry was performed using a standard protocol [20 (link),21 (link)]. In brief, mice were anesthetized with an i.p. injection of Nembutal (pentobarbital sodium, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) at 0.5 mg/kg and transcardially perfused with phosphate-buffered saline (FUJIFILM Wako), followed by 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako). The brains were immersed in 4% paraformaldehyde for 24 h before being transferred to a 30% sucrose solution (FUJIFILM Wako) for 24 h. Coronal brain sections of 40 μm thickness were cut using a cryostat (Carl Zeiss MicroImaging GmbH, Jena, Germany) after the brains were rapidly frozen in OCT compound (Sakura Finetek, Torrance, CA, USA). Microglia were detected in the prefrontal cortex (PFC) and hippocampus slices dissected from frozen brains using the rabbit polyclonal anti-mouse ionized calcium-binding adapter molecule 1 (Iba-1) antibody (FUJIFILM Wako, 019-019741). Microglial TNF-α expression was confirmed using the goat polyclonal anti-mouse TNF-α antibody (R&D Systems, Minneapolis, MN, USA. AB-410-NA). The secondary antibodies used were Alexa Fluor™ 488-conjugated anti-rabbit IgG (1:300; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor™ 594 anti-goat IgG (1:300; Invitrogen). The nuclei in the slices were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Cell images were acquired using a fluorescence microscope (Axio Scope.A1; Carl Zeiss, Oberkochen, Germany). The levels of Iba-1 signals were obtained using ImageJ 1.53K software (NIH Image, Bethesda, MD, USA).