Axiovs 40 4

Male C57BL/N mice (25 to 30 g) were randomly separated into two groups (n = 6/group). One group was administered cocaine (20 mg/kg, IP) daily for 7 days and sacrificed 1 h after the final injection. Mice similarly treated with 0.9 % saline of the same volume served as controls. Animals were transcardially perfused with the fixative, and immunohistochemical procedures were performed as described below. Floating tissue sections (30-μM-thick) were co-incubated with primary anti-mouse ionized calcium-binding adapter molecule 1 (Iba1) (Abcam, Cat# ab15690), anti-rabbit TLR2, anti-goat Iba1 (Abcam, Cat# ab5076), and anti-mouse CD68 antibody (Dako, Cat# M0814) overnight at 4 °C. Alexa Fluor 488 conjugated anti-mouse or anti-goat (Life Technologies, Cat# A11001; Invitrogen, Cat# A11055) and Alexa Fluor 594 goat anti-rabbit secondary antibodies (Invitrogen, Cat# A11008) were added for 2 h to detect Iba1 and TLR2 followed by mounting of sections with DAPI (Invitrogen, 36935). Fluorescent images were acquired at room temperature on a Zeiss Observer Z1 inverted microscope (Carl Zeiss, German); images were processed using AxioVs 40 4.8.0.0 software (Carl Zeiss MicroImaging). Photographs were acquired using an AxioCam MRm digital camera (Carl Zeiss, German).

Animals were perfused, and immunohistochemical procedures were performed as described later. Rapidly frozen sections with 20 μM were co-incubated with primary anti-AIF1 antibody (Wako Pure Chemical Industries, Chuo-ku, Osaka, Japan, 019-19741) and anti-LAMP2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, sc-19991) overnight at 4°C. Secondary AlexaFluor 488 goat anti-rabbit IgG (A-11008) or AlexaFluor 594 goat anti-mouse (A-11032) from Thermo Fisher Scientific Waltham, MA, USA, was added for 2 h to detect Iba1 and LAMP2, followed by mounting of sections with prolong gold antifade reagent with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA, USA, P36935). Fluorescent images were acquired on a Zeiss Observer. AxioVs 40 4.8.0.0 software (Carl Zeiss, Thornwood, NY, USA) was used to process the images.