Nonsynaptic GluRIIA and GluRIIB protein is intracellular. (A) 3D confocal reconstruction of ventral longitudinal muscle (VLM) 6 and 7 from a 24BGal4/UAS–dicer-1.RNAi larvae. Before fixation, the muscle plasma membranes were stained by FM1-43FX (red). In the NMJ (arrows), GluRIIA (green) and GluRIIB (blue) immunoreactivity is visibly associated with the plasma membrane. However, as shown by the “cut-away” plasma membrane section of VLM 6 (bottom left), most of the GluRIIA and GluRIIB immunoreactivity is intracellular and obscured by the membrane staining. (B) 3D reconstruction of the ventral portion of another VLM 6 and 7, viewed in cross section to show abundant intracellular GluRIIA and GluRIIB protein (gold). Bar, 10 µm. (C) Confocal stack of the images used to create the image in A. (D) 3D reconstruction, as in A, showing only muscle plasma membrane (red) and GluRIIA (green). Note the GluRIIA protein associated with the NMJ. (E) Same data shown in D, but with the red muscle membrane not shown. Note the abundant GluRIIA that was previously obscured by the plasma membrane, which is consistent with the nonsynaptic GluRIIA being intracellular.