Diaminobenzidine dab peroxidase substrate kit

For Iba-1 labeling, mice were perfusion-fixed with 4% formaldehyde. Brains were dissected, postfixed in 4% formaldehyde for 24 hrs and immersed in 20% sucrose for another 24 hrs at 4°C. Brains were frozen at −80°C. Later, 40-μm-thick coronal sections were cut and collected with a cryostat. Sections were then incubated in 1% sodium borohydride and 0.5% hydrogen peroxide to reduce background. They were incubated with anti-Iba1 primary antibody (1:1000; rabbit anti-mouse, Cat# 019-19741; Wako Chemicals, Richmond, VA), followed by Alexa Fluor 594 conjugated secondary antibody (1:500; ThermoFisher Scientific, Grand Island, NY).
For COX-2 and c-fos labeling, fresh brains were rapidly frozen in −80°C isopentane for 20 sec and 20-μm-thick coronal sections were later generated with a cryostat. Sections were fixed in an acetone/alcohol mixture, followed by incubation in a glucose oxidase and sodium azide solution. They were incubated with anti-COX-2 (1:200; rabbit-anti-mouse, catalog #160106; Cayman Chemical, Ann Arbor, MI) or anti-c-fos (1:1000; rabbit-anti-mouse, catalog #sc-52; Santa Cruz, Dallas, TX) primary antibody and anti-rabbit/anti-rat secondary antibody. The labeling was amplified using an ABC solution (Vector Laboratories, Burlingame, CA) and visualized with a diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Burlingame, CA).