Immunohistochemical Analysis of Neuroinflammation

Briefly, after mice were anesthetized with isoflurane and intracardially perfused with ice-cold PBS and 10% formalin, the brains were removed, fixed in 10% formalin overnight at 4 °C, and dehydrated with 30% sucrose for 3 days. Brain tissues were snap-frozen at − 80 °C and cut into 10-μm-thick coronal sections using a cryostat (CM3050S; Leica Microsystems, Bannockburn, III, Germany). Immunofluorescence staining was performed as previously described [7 (link)]. Brain samples were incubated overnight at 4 °C with primary antibodies including rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, Abcam), rabbit anti-glial fibrillary acidic protein (GFAP, 1:100, Abcam), rabbit anti-neuronal specific nuclear protein (NeuN, 1:200, Abcam), rabbit anti-myeloperoxidase (MPO, 1:500, Abcam), and goat anti-TREM2 (1:1000, Abcam). The sections were then incubated with corresponding secondary antibodies (1:200, Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature, followed by visualization using a fluorescence microscope (Leica Microsystems).