Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Thaw a section of frozen rat brain tissue.

Add buffer and mince the tissue. Then, transfer it to a tube containing buffer and mix.

Centrifuge the mixture and discard the supernatant.

Add a proteolytic enzyme solution to the tissue and resuspend it using a large-diameter pipette.

Incubate the mixture to break down the extracellular matrix, loosening the neural cells.

Centrifuge the mixture and discard the supernatant.

Resuspend the tissue fragments in a chilled buffer and pipette them repeatedly to dissociate the cells.

Allow the debris and undissociated cells to settle, then transfer the single-cell suspension to a new tube.

Transfer the cells into ice-cold ethanol and mix. Incubate them to fix and permeabilize the cells.

Centrifuge the suspension and discard the supernatant.

Resuspend the neural cells in a cold buffer for further analysis.

To mince the tissue, allow the samples to thaw on a cold glass plate for no more than 1 minute before covering the samples in one to two drops of buffer A solution. Next, holding a razor blade completely vertical to the plate, mince the tissue 100 times in each orthogonal direction. To thoroughly mince the tissue, it is critical to keep the tiss