Fig. 3.

MGL labels presynaptic terminals. HEK293 cells were transiently transfected with V5-tagged rMGL and stained using V5 (1:500) and MGL (1:2000) antibodies. A, DAPI staining identifies cell nuclei (arrowheads). B, V5 antibody (detected with Texas Red secondary) staining shows two V5-rMGL transfected cells (nuclei indicated by arrows). C, mMGL staining (FITC) labels the same cells identified with the V5 antibody. D, Overlay of V5 and mMGL images shows both antibodies overlap completely, DAPI fluorescence identifies all cells in the field. The nontransfected cells are not labeled with the mMGL antibody. Scale bar, 15 μm. E, left, MGL staining of an autaptic culture; right, staining (taken at same settings) when the MGL antibody was coincubated with immunizing protein (IP, 5 μg/ml). Scale bar, 25 μm. F, separate images from same cell show on the left (green) SV2, a marker for presynaptic terminals, and on the right MGL (red). Arrows identify two of the numerous points of overlap. Scale bar, 10 μm. G, left, MGL (red) and staining for axonal marker 2H3 (green, overlap in yellow). Right, MGL (red) and 2H3 (green) channels, with arrows identifying two points of overlap. Scale bar, 12.5 μm. H, left, MGL (red) and staining for CB1 (green, overlap in yellow). Center, MGL. Right, CB₁, with arrows identifying two points of overlap. Scale bar, 12.5 μm.