Immunofluorescence Staining of Frizzled-7 in Brain

We performed double immunofluorescence staining as previously described [3 (link)]. The whole brains were fixed in 4% paraformaldehyde overnight followed by the dehydration in 20% sucrose solution and 30% sucrose solution. The brain specimens were stored − 20 °C until use. The brain specimen was cut into 10 μm thick coronal sections with a cryostat (CM1860; Leica Microsystems, Germany). Immunofluorescence staining was performed with the following primary antibodies: rabbit anti-frizzled 7 (1:100; ab64636, Abcam, MA), goat anti-frizzled 7 (1:100; # PA5-47232 Thermo Fisher, MA), goat anti-glial fibrillary acidic protein (GFAP) (1:200; ab53554, Abcam, MA), and mouse anti-neuronal nuclei (NeuN) (1:100; ab104224, Abcam, MA), and mouse anti-von Willebrand Factor (vWF) (1:100; sc-365712 Santa Cruz Biotechnology, TX. After the secondary antibody incubation, the brain sections were visualized and photographed with a fluorescence microscope (Leica Microsystems, Germany). Adobe Photoshop software was used to analyze the microphotographs.