Immunostaining of Brain Sections

Tissue processing and immunostaining were performed on free-floating sections (40 µm) following standard published techniques.^{13 (link)} Coronal brain sections were incubated overnight with either rabbit anticalbindin (1:15 000–1:100 000; Swant), biotinylated rabbit anti-3D6 (1:3500; Elan Pharmaceuticals), or mouse antisynaptophysin (1:1000) primary antibodies. Calbindin and 3D6 staining was revealed using the ABC kit (Vector) with diaminobenzidine (Sigma-Aldrich). Mean signal intensity was measured using National Institutes of Health ImageJ software. Synaptophysin was labeled with a fluorescent secondary antibody and quantified as previously reported.^{14 (link)}