I would like to quantify the expression of a protein in neurons. However, it is also highly expressed in glia. How can I exclude the glia protein expression? I mean to test the protein levels specific in neurons. Methods using culture neurons or the whole animal are both OK for me.
Thanks a lot.
You could perform Laser Capture Microdissection on slides of tissue to selectively collect micro-quantities of neurons and then select a very sensitive protein detection method.
Or, why not trying the Duolink kit for in situ PLA? http://www.olink.com/products/duolink
You can sort cells with flow cytometry and measure your protein concentrations in just the neuron-enriched cell population. This would work well if your proteins of interest are nuclear.
http://www.ncbi.nlm.nih.gov/pubmed/20717962
I suggest to look into the Banker cell culture method for hippocampal neurons, which is said to provide very pure neuronal cultures, with little contamination from glia cells: http://www.ncbi.nlm.nih.gov/pubmed/?term=banker+2006+nature+protocols
The idea is this: you plate glia cells in a dish or well and let them grow for a week, then you prepare embryonal hippocampal cultures under conditions that kill off glia contamination and seed the neuronal progenitor cells onto a cover slip on which you attached little "feet" of wax or another inert substance, and then you flip the cover slip upside down onto the prepared dish seeded with glia. This culture method is also known as "sandwich culture". The factors secreted by the glia are essential to keep the neurons alive and healthy and support their maturation, but the glia cells do not actually come into contact with the neurons in this culture method. Once the desired age and density of the neurons is reached, you simply take out the cover slip and harvest the neurons.
The downsides of the Banker culture method are its complexity and potential qualitative heterogeneity. I suggest to get into contact with a specialist at your institute.
The problem I see with microdissection is that it is probably not possible to separate glia and neurons in a mature brain. They are just too closely intermingled.
FACS, unfortunately, will not work on whole neurons because they are too fragile.
Thanks all for your suggestion. For the sandwich culture methods, can I growth glia on the bottom of 24 well plate and add FDU or 4Ara-C with the coverslips carrying target neurons? Therefore, division of new glia on the coverslip will be inhibited. @Sven Truckenbrodt Is it OK if I intermingle some neurons with glia in the supporting layer?
Anyway, thanks a lot.
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