Dear Behnam,

I have a couple of questions regarding your desired imaging protocol.  Just to be clear,  do you want to label the intracellular portion of the membrane, or the extracellular portion?

Is there some reason you can't fill your neurons with something like Lucifer Yellow after recording?

Thanks--

Jill

Hi Jill, 

I'm trying Expansion Microscopy using conventional antibodies, so I'm worried that the lack of a membrane protein binding partner in the biocytin-streptavidin-Alexafluor filling  dye may result in a loss of fluorescence. I intend to use a fluorescent protein to bind neurons via intracellular filling to isolate single neurons in mouse cortex, then use the expansion microscopy protocol to digest the tissue but preserve some of the fluorescence that I can later reconstruct with software to analyze spines. Does that make sense? 

Thanks - Ben

Dear Behnam,

I would suggest to use widely available GFP (Green fluorescent protein) that is widely available. In the past I have filled neurons with GFP or GFP-conjugated molecules, and in your case should not be too difficult to fill cells with GFP in whole-cell configuration. Addition of some extra electrical pulses generated by your amplifier should ease the passage of large molecules (See below for an example).

Best wishes, Refik

Article Semi-loose seal Neurobiotin electroporation for combined str...

Dear Behnam,

Thanks for the explanation.   As Refik has pointed out, you can fill with GFP-conjugates.  I had thought of Lucifer Yellow simply since it is usually easy to fill the neuron with it, and there is (I think; haven't checked if it is still available) an antibody to it so that you could enhance the signal after fixation.  The same is, of course, true for GFP or YFP conjugates.

Good Luck!

Jill

Hi Jill and Refik,

As far as I know, GFP doesn't bind to anything with the neuron, it just floats around (please correct me if I'm wrong). Preferably, whatever I injected into the cell will find a binding partner. This could be an antibody that I can then stain using a secondary. 

Ben

Dear Benham,

So, if I understand you correctly, you are not going to use thel probes that the Boyden group designed for Expansion Microscopy?  They have one that works with the mice that express YFP in a subset of neurons, and that YFP is not bound to anything.  It is just expressed selectively in the neurons from a Thy1 promoter.

http://syntheticneurobiology.org/PDFs/15.01.chen.FULL.pdf

Unfortunately, I study axons and not dendrites.  All of the proteins that I can think of off hand that are found on the intracellular side of dendrites are either only in the dendritic shaft, or are only in spines. Maybe something that targets actin might get you the structure of dendrites and spines?

Sorry to be so unhelpful.

Cheers!

Jill

Thanks Jill, I think that I may be able to use an intracellular fluorophore and perhaps that may work.