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Fig. 4. Confocal images of a triply labeled NMJ. The sternomastoid muscle of
a live animal was first labeled with BTX-biotin/strept-488 (green), then 3
days later with strept-594 (red) to label recycled receptors. Two days after
recycled receptor labeling, the animal was perfused with 2% PFA, the
sternomastoid muscle sectioned and immunostained with anti-AChR. Green arrows
indicate accumulation of green fluorescent streptavidin that has been removed
from pre-existing AChRs after internalization, which may correspond to
degradative vesicles. Yellow arrows indicate accumulation of green and red
streptavidin tags removed from pre-existing and recycled receptors after
internalization, which probably also correspond to degradative vesicles. Blue
arrows indicate vesicles containing AChRs, but lacking any streptavidin-Alexa
tags; these vesicles may correspond to internalized AChRs in later stages of
recycling and/or degradation, or newly synthesized receptors in the process of
insertion. White arrows indicate vesicles containing receptors and their
streptavidin tags after internalization. Colors in the overlay have been
adjusted in Photoshop (Adobe) to maximize contrast. Boxed areas (above) are
enlarged below. The results suggest that the vesicles containing fluorescent
products (yellow arrows) may be stable, whereas those corresponding to cycling
AChRs (white arrows) may be more dynamic. Scale bar, 20 µm.
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