VGLUT immunoreactivity colocalizes with vesicular marker synaptobrevin 2. A, Astrocytes were identified based on their ability to intracellularly accumulate β-Ala-Lys-N_ϵ-AMCA (blue). Double labeling using indirect immunocytochemistry against VGLUT 2 (B; red) reveals punctate immunoreactivity that colocalizes with β-Ala-Lys-N_ϵ-AMCA (C; overlay of A and B; pink indicates colocalization), as shown in X-Z (C; bottom) and Y-Z (C; right) projections from the regions identified by dotted lines. Double labeling of astrocytes with synaptobrevin 2 (D; green) and a mixture of antibodies labeling all three isoforms of VGLUTs (E; red) indicates that VGLUT immunoreactivity substantially overlaps with vesicular marker synaptobrevin 2 (F; overlay of D and E; yellow indicates colocalization). Scale bars: C, X-Z and Y-Z projections, 5 μm; A–C, 10 μm; D–F, 20 μm.