If one uses antibodies against VGLUT1 and synaptophysin to stain in WT cultured hippocampal neurons, one expects the immunosignals to co-localize, since both are synaptic vesicle specific proteins. how to explain the finding of solely vglut1 signals? I understand if I have only synaptophysin signals, maybe I thereby detected vgat expressing neurons, but synaptic vesicles that are positive for vglut1 and not stained with anti synaptophysin antibody?
To be sure, both primary and secondary antibodies do not show cross reaction? Which antibodies do you use? Do both antibodies work when applied in a single staining?
Thank you two for your answers ! So in principle Dirk is right. I see coloc's as well as single green and/or red spots. Interestingly, the colo's are also "only" partly overlapping, not completely. Must be an artifact, since permeabilizing and blocking neurons in culture at +4°C reduces the single spots and elevates the number of spots with 100% coloc.
well more simply : may be synaptophysin is not in all sv. May be sv are heterogeneous ? And therefore there are some vesicle that are VGLUT1-positive and synaptophysine-negative in some vesicular pools ?
I am not surprized by negative syn terminals
for example VGLUT2 and VGLUT3 (on which we work) very poorly colocalize with syn
and only half a gaba terminals are syn-positive
with VGLUT1 I would estmate that around 20% of VGLUT1-positive terminals are syn-negative
How to explain it ?? I don't know for sure
1) if you look at these synaptic proteins they are heterogeneous not only between synapses but also inside synapses
2) as far as I know the role of synaptophysin is not clear
3) and we do not have a generic marker of all synpases
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