This article describes a method for isolating nuclei from fresh frozen human brain tissue using mechanical and centrifugation techniques. The process involves the use of lysis and sucrose buffers to create a density gradient for effective separation.
Transfer a piece of fresh frozen human brain tissue to a suitable grinder containing cold lysis buffer.
Move the pestle up and down to mechanically break the tissue, releasing the cells.
The detergent molecules in the lysis buffer break open the cell membrane, releasing the nuclei.
Transfer the contents to a suitable centrifuge tube and add cold sucrose buffer to the bottom of the tube, creating a density gradient.
Centrifuge at high speed to separate the nuclei at the bottom of the tube.
Discard the supernatant and resuspend the nuclei in a buffered saline solution.
Add target-specific fluorophore-conjugated antibodies to the nuclei and incubate to allow the antibody to bind to its target nuclear protein.
Add nuclear staining dye to visualize the intact DNA.
Perform fluorescence-activated nuclei sorting to sort the nuclei of different cell populations based on distinct fluorescence signals.
Begin dissecting approximately 200 to 400 milligrams of tissue from a fresh, frozen human adult cortex tissue sample. Place the tissue in a 7-milliliter glass tissue Douncer containing 4 milliliters of ice-cold lysis buffer on ice, and grind the tissue approximately 50 times.
Transfer the homogenate to a 12-milliliter ultracentrifuge polypropylene tube. Use a 5-milliliter pipette to add 6.5 milliliters of ice-cold sucrose buffer to the bottom of the ultracentrifuge tube without disturbing the interface between the sucrose and the tissue homogenate.
To collect the nuclei, ultracentrifuge the lysate for one hour at 101,814 times g, and carefully aspirate the supernatant and debris without disturbing the pellet. Add PBS to the ultracentrifuge tube, resuspending the nuclei pellet.
For fluorescence-activated sorting of the isolated nuclei, add approximately 20 microliters of the resuspended sample to an antibody solution containing AlexaFlour555-conjugated mouse anti-NeuN antibody. Add approximately 20 microliters of the resuspended sample to an antibody solution containing APC-conjugated mouse anti-PAX6 antibody. After a one-hour incubation, at 4 degrees Celsius with shaking protected from light, add DAPI at a 1 to 1,000 ratio to all of the samples and controls.
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