FIGURE 3.

Isolation of nuclei from frozen brain samples. Human postmortem frontal cortex was dissociated; nuclei were isolated and stained with the nuclear stain DAPI and a monoclonal NeuN antibody conjugated to PE. The panels represent an example of a single sort experiment performed using a particular brain donor (subject ID 1690). (A) Nuclei were first sorted based on their forward and side scatter from all possible events (R1 gate). (B) Single nuclei were further sorted based on their size from the doublets or larger clumps of nuclei (R2 gate). (C) DAPI positive single cells were gated as either NeuN-PE positive (neurons, R3 gate) or NeuN-PE negative (glia, R4 gate). (D) Post-sort data showing the purity (>97%) of the separation between neuronal and non-neuronal nuclei. (E) Fluorescence image showing unsorted nuclei stained for NeuN (green) and DAPI (blue). The scale bar represents 10 μm. (F) Box-and-scatterplot chart of the ratios of neuronal (NeuN+) and non-neuronal nuclei (NeuN-) from total nuclei for each neuropathological group. Each point represents the ratio for an individual sample. The box denoted the mean (thick horizontal line) and upper and lower quartiles (thin shorter lines).