Is there anybody that has some experience with FM1-43 staining?
I am trying to label synaptic vesicles in cultured hippocampal neurons
using the following protocol:
(a) 90sec -- 10 uM FM1-43 with Hi K+
(b) 90sec -- 10 uM FM1-43 with standard solution*
(c) 3min -- standard solution with 1uM ADVASEP-7
(d) 40min -- standard solution
* Note that the standard solution contains DNQX, Kynurenic acid, 0.2mM
Ca2+ and 5 mM Mg2+ (to reduce spontaneous release).
During the 40 min of step (d) i take a picture every 4 min.
I got told that i could expect a sudden drop of intensity within the
first 1-3 minutes (so between the the first two images) and then a
stable (at least for the following 20-30 minutes) baseline. But what i
observe is a constant exponential decrease of fluorescence intensity,
that doesn't reach a stable plateau during the 40 min window.
Can this be due to photobleaching? As light source i used a bright
mercury lamp with a short exposure time (50ms) and dimmer LEDs with
500 ms exposure time. Both approaches resulted in the same outcome.
So, if this effect is due to photobleaching, shouldn't these two
conditions result in different kinetics?
Does anybody have an idea for this problem? In detail: is it actually
possible to get such a baseline?
Any comments/suggestions concerning the protocol?
All comments are appreciated!