We want to perform image analysis to characterize neuron branching, post patch clamp experiment, in fixed neurons.
I’ve had success using non-reactive Alexa dyes.
Here‘s one: https://www.thermofisher.com/order/catalog/product/A33077
Alexa Fluors will indeed work, but depending on your fixing and processing protocol, you might find them substantially quenched.
In the past I have used low molecular weight dextran conjugates, that are tagged with a fluorophore and a biotin moiety. These labels diffuse fairly quickly, are fixable, fluoresce on their own and the fluorescence can later be enhanced by using streptavidin tags during the tissue processing. I used this to reconstruct cerebellar parallel fibers, and the labeling was great.
Invitrogen/Thermo offer compounds like this, e.g. MicroRuby:
https://www.thermofisher.com/order/catalog/product/D7162?SID=srch-srp-D7162
If you want to go more classic, try luciferyellow. It fills cells nicely, is fixable and stays bright after fixing. You will need fairly high concentrations, though, so the lithium salt (rather than potassium) would make sense. Do keep in mind that lithium interferes with slow calcium signalling by modulating enzymes in the IP3 pathway, so if that is what you are interested in, LY is a no-go.
Good luck!
I would highly recommend 3-4% of neurobiotin in the patch pipette. After fixation and processing with streptavidin you will be able to see clearly not only the somas but also processes.
Alexa dyes should work well.
Article Retinal Waves Modulate an Intraretinal Circuit of Intrinsica...
One thing to keep in mind with Neurobiotin and Alexa dyes is that they will cross gap junctions. Depending on your cell of interest that might be an issue. But it doesn't have to be.
Regarding the bleaching of Alexa Fluors during fixation, at least for some of the common Alexas, antibodies are available that can be used to boost the signal, if you find that you do not get sufficient labeling intensity.
Christian makes a good point abot crossing NB gap junctions. However, from our experience NB labeles predominantly somas of coupled cells.crossing
I have only ever used Lucifer Yellow, but that is because I am "old school". However, here is a link to another researcher here on RG that had good luck with neurobiotin:
Article Semi-loose seal Neurobiotin electroporation for combined str...
Good Luck!
Jill
1.5% A568hydrazide works the best in my hands. It's not Gj permeable.
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