Neuronal nuclei isolation from human postmortem brain tissue.
Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan. Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays.
Matevossian
Anouch
A
Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School, MA, USA. Anouch.Matevossian@umassmed.edu
Akbarian
Schahram
S
D001921:Brain / Q000166:cytology / Q000648:ultrastructure*; D001923:Brain Chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods*; D005629:Frozen Sections; D006801:Humans; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure*; D011180:Postmortem Changes; D014461:Ultracentrifugation / Q000379:methods*