I am trying to find protocols for the isolation of neuronal cells from different regions of the brain, so far I have found only a few that deal with either different areas of the brain such as the cortex, or the spinal cord. Or perform isolation in post-natal (1-2 days), or embryos. I don't know if it the same protocol for all neurons regardless of the age of the mice or brain region. I never have done isolation of neurons before, and I am not a neurobiologist either. If someone could share a protocol with me and give me some advice on how this is done, I would deeply appreciate it.
I found and answer kind of, I wrote this small protocol maybe it will help
Isolation of neuronal cells will be done in mice strain C57BL/6 mouse strain of the brains of postnatal day (1-2) mice. Dissection and dissociation of the brain was used as described previously97,98. Briefly, the cerebral cortex, cerebellum, thalamus, and brain stem will be dissected from the brain and dissociated by papain (Worthington Biochemical, LS03126) digestion for 1 h at room temperature, followed by mechanical disaggregation with a Pasteur pipette. Cell will be the plated in a 24-well plate at 150,000 cells/well pre coated with 0.01% poly-D-lysine (Sigma-Aldrich, P7280) and 10 μg/mL laminin dissolved in PBS. Neuronal cultures will be maintained in NeurobasalTM-A Medium (Invitrogen, 1088022), supplemented with B27 supplement (Invitrogen, 17504-044), Glutamax, antibiotics (100 U/Ml penicillin, and 100 μg/mL streptomycin), and mitotic inhibitors (10 μM uridine + fluoro-deoxyuridine). Cultures will be fed every other day by replacing half of the media with fresh, complete media. Neuronal primary cells will be infected with different MOI’s WNV and WNV collection will be done every 8 hours, finishing at 72 hours with collection of cells for immunoblotting.
97. Hilgenberg, L. G. W. & Smith, M. A. Preparation of dissociated mouse cortical neuron cultures. J. Vis. Exp. 4–7 (2007). doi:10.3791/562
98. Sierra-Fonseca, J. A. et al. Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction. BMC Neurosci. 15, 1–19 (2014).
I was told the mice needed to be that age of the neurons will die out.
However, if what you are planning is find the expression of proteins or transcript is just better to do immunohistochemistry of brain slides.
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