Figure 1. Whole-mount immunostaining of the TVA muscle for reliable detection of antigens at the NMJ. A, The flowchart depicting the protocol used for clean and reliable immunostaining of muscle fibers. B, Example images of the immunostaining. Top left, A low magnification image shows NMJ innervation patterns along the TVA muscles. Integrin α7 (white), used to highlight the membrane of individual muscle fibers, was colabeled with AChRs (green) to highlight the innervation pattern along the TVA muscle. Scale bar, 100 µm. Top right, A high magnification image of an individual NMJ shows the pretzel-shaped AChR distribution at the NMJ of a TVA muscle. Note that AChRs are not uniformly distributed. Scale bar, 5 µm. Bottom: AChRs (green) were colabeled with tubulin (white), an intracellular antigen, to show that the whole-mount method enables excellent antibody penetration to label the microtubule network inside the skeletal muscle. Scale bar, 20 µm.