A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus
Transkript
Take dentate gyrus from a mouse brain in a cold homogenization buffer.
Mechanically dissociate to release neurons, glia, and stem cells.
The buffer's mild detergent permeabilizes the cells, releasing nuclei and other organelles.
Filter the homogenate to remove tissue debris. Then, centrifuge, remove the organelles, and resuspend in the homogenization buffer to lyse any intact cells.
Centrifuge again, remove the supernatant containing released organelles, and resuspend the nuclei in a wash buffer.
Filter to remove any debris, centrifuge, and remove the supernatant.
Introduce a fluorescent DNA stain to label all nuclei and a fluorophore-conjugated antibody that binds to the neuronal nuclear antigen, or NeuN, labeling the neuron nuclei.
Using fluorescence-activated nuclei sorting, identify aggregates with higher DNA stain fluorescence intensity and higher scattering properties, separating the single nuclei.
Analyze the single nuclei, separating the NeuN-positive neuronal nuclei and isolating the NeuN-negative nuclei belonging to the glia and stem cells.
