I would like to analyse mRNA and different isoforms of a protein (but not necessary at the same time) in neurons, astrocytes and microglia from a brain of adult mouse. (I have no antibodies to detect the different specific isoforms...). What could be the best option ? In situ hybridation with one probe against isoforms of interest and another one specific to the different cell types?
Dear Marie A lot of work! indeed !!
If I understand well your objective you may do double in situ/immuno labeling, in situ to detect the mRNA of the isoform and immuno to label cell type. The technique is detailed in this paper.
Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis.
Lanciego JL, Barroso-Chinea P, Rico AJ, Conte-Perales L, Callén L, Roda E, Gómez-Bautista V, López IP, Lluis C, Labandeira-García JL, Franco R.
J Psychopharmacol. 2011 Jan;25(1):97-104. doi: 10.1177/0269881110367732. Epub 2010 May 20.
Agree with Rafael.
In addition, you can separate these cells by density gradient centrifugation and then culture them, but the purity is not guarantied.
Thanks you a lot for your answers. Dear Yuxiang, Do you talk about cells from adult brain? Do you manage to culture them ?...I say that because if we are talking about immature cells, I could use the very useful and friendly website : http://web.stanford.edu/group/barres_lab/brain_rnaseq.html , but it looks like this has been made with cortex from P1-mice...,Actually I am interested in cells from adult brain. It is likely that the expression of the protein that I want to study would be different between post-natal days and at adult age...
I dont believe that centrifugation help to separate and maintain a viable the cells
Hi Marie,
Yes, I thought of this because I did adult neuron culture, and in one of the gradient step the microglia is in the bottom, while oligodendrocyte and neuron in upper fractions. You can refer to this paper:
Brewer, G.J., Torricelli, J.R., 2007. Isolation and culture of adult neurons and neurospheres. Nature Protocols 2, 1490–1498. doi:10.1038/nprot.2007.207
BTW, if you want to do adult neuron culture, keep in mind that your target protein expression may be different in your culture from in vivo condition.
The above Nature protocol does work (I've used it), but be aware that there is a mistake in the optiprep osmolality measurement. You'll need to fix that for the protocol to work robustly...
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