A two-color method of bungarotoxin labeling of AChRs combined with nuclear labeling identifies fibers where the muscle segment underneath the NMJ has degenerated and regenerated and altered the synapse. Alexa 647 bungarotoxin (first color) was applied vitally at a nonsaturating concentration to the sternomastoid muscle. Two weeks later, the muscle was dissected, fixed, labeled with rhodamine–bungarotoxin (second color) and with DAPI, and imaged by confocal microscopy. Confocal z-slices (far right images) were prepared in each case and the contrast and brightness manipulated so that outline of the muscle fiber could be ascertained as well as a fiber nucleus (asterisk) and AChRs (arrowheads). A, B, Components of each of two junctions from the same 25.5-month-old muscle: first-color AChRs, second-color AChRs, nerve terminals, and nuclei (DAPI). A, Young-appearing junction with strong first-color labeling whose fiber nuclei are at the periphery. B, Junction whose first color labeling is much weaker, whose fiber has a string of central nuclei (arrows) and whose AChRs have become fragmented. Each asterisk in the en face images identifies the same nucleus shown with an asterisk in the z-section. The terms “strong” and “weak” in reference to the first color label are relative to each other, as explained in the text. Scale bar, 10 μm.