Fig. 3. Fluorescent tags from recycled receptors are endocytosed and targeted to vesicles containing strept-488 from pre-existing AChRs. The sternomastoid muscle was labeled first with BTX-biotin/strept-488, then 3 days later with strept-594 to label recycled receptors. (A) Recycled and pre-existing AChRs imaged 2 days after recycled receptor labeling. Note that all the vesicles containing red fluorescence from recycled AChRs also have green signals from preexisting AChRs (arrows), whereas some of the green spots lack red signal (arrowheads). (B) A NMJ was labeled as in A and then imaged periodically over the next 28 hours. Note that a number of the vesicles containing strept-488 from the pre-existing AChRs at time 0 are stable over the next 28 hours (arrows), whereas others are transient. The strept-594 labels from recycled receptors are selectively targeted to the stable vesicles containing fluorescent label from pre-existing receptors. Scale bars, 20 µm.