Immunofluorescence Staining of Mouse Brain

Mice were perfused under deep anesthesia with ice-cold PBS followed by perfusion with 10% formalin at 24 h after surgeries. The brains were removed and fixed in formalin at 4 °C overnight and dehydrated with 30% sucrose for 3 days. Brain samples were then snap-frozen at − 80 °C and cut into 10-μm-thick coronal sections using a cryostat (CM3050S; Leica Microsystems). Immunofluorescence staining was performed as previously described [36 (link)]. Briefly, brain samples were incubated overnight at 4 °C with primary antibodies including goat anti-Iba-1 (1:100, Abcam), goat anti-GFAP (1:100, Abcam), goat anti-NeuN (1:200, Abcam), rabbit anti-MC4R (1:500, Abcam), and rabbit anti-MPO (1:500, Abcam). The sections were then incubated with corresponding secondary antibodies (1:200, Jackson Immunoresearch, West Grove, PA) at room temperature for 2 h and followed by visualization using a fluorescence microscope (Leica Microsystems, Germany).