Identification of VGluT2-IR and CGRP-IR afferents in lamina I using 3D co-localization with Neurobiotin labeling. A: Montage of maximum projections of confocal image stacks (11 images per stack; z-interval = 1 μm) showing primary afferents labeled with Neurobiotin (green) and terminals labeled for VGluT2-IR (magenta) and CGRP-IR (red). Representative locations for high resolution analyses of medial, mid and lateral regions are indicated by white boxes. B-D: maximum projections of high resolution confocal image stacks (41 images per stack; 80 × 80 μm; z-interval = 0.5 μm) from medial (B), mid (C) and lateral (D) regions of lamina I (boxed areas in A). E-G: 3D reconstructions of data from image stacks in B-D, respectively, showing all Neurobiotin-labeled fibers (green), VGluT2-IR terminals (purple) and CGRP-IR terminals (red). H-J: 3D colocalization showing only Neurobiotin-labeled terminals containing VGluT2-IR (blue) or CGRP-IR (yellow). Scale bar represents 100 μm in A. Scale bar in D represents 10 μm and applies to panels B-J. LT; Lissauer's Tract, I; lamina I, IIo; outer lamina II.