Mutant VAPB inclusions are localized in the RER and consist of smooth ER-like tubular profiles and electron dense material. A-D) Transmission electron photomicrographs of mutant VAPB inclusions in spinal motor neurons of a 6 weeks old VM1 (A, B), a 6 week old VM3 (C), and a 70 weeks old VM1 x VM5 mouse (D). Note in A that the inclusions (red arrows) are localized within normal appearing rough ER (RER). Other organelles such as Golgi apparatus (Go) and mitochondria (m) are unaltered (A, B). In C the ER lumen is accentuated in green to outline the continuity of tubular smooth ER like profiles in the inclusions with the surrounding rough ER cisterns. Note in C and D that the electron dense material in the inclusions has a patchy appearance (red arrows). E) Post-embedding immunogold electron microscopy with anti-HA antibody showing that HA-labeling is preferentially associated with the electron dense material in the inclusions (see insert). The dashed lines outline two distinct inclusions. Color overlay in A: red, area outside the motor neuron; yellow, nucleus; green, lumen of ER. Scale bars: 2 μm (A), 500 nm (B), 200 nm (C, D, E).