Question to FM1-43 staining of synaptic vesicles

[Tom]

Hi!

Does anybody have some experience with FM1-43 dyes, especially when
used for staining of synaptic vesicles?

I am using FM1-43 for staining of synaptic vesicles in primary
hippocampal cultures (via 600 APs @ 10 HZ). Subsequently i want to
measure synaptic release by applying 2 electric pulses (1st: 40AP
followed by 2nd: 600AP) and monitor the resulting fluorescence
decrease of synaptic spots.
After a first look, my results seemed alright and i found my
everything in accordance with the data one can find in literature. But
a second look got me thinking. Spots i picked away from synapses (on
soma or dendrites) showed a pattern of destaining that was similar to
the one of the synapses, even though absolute values were different.
But more confusing to me, the ratio of destaining after 40APs /
destaining after 600AP, which should be an estimate of the readily
releasable pool size was identical in synaptic spots, soma/dendrite
spots, and even in background pixels!

Does anybody have an idea how to explain this confusing result?

Thanks for any ideas!

Thomas