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. Author manuscript; available in PMC: 2021 Aug 30.
Published in final edited form as: J Vis Exp. 2021 Apr 16;(170):10.3791/62405. doi: 10.3791/62405

Figure 3: Dependent and independent metrics for successful FANS experiments.

Figure 3:

(A) Effect of sample collection PMI on the quality of nuclei: lower PMI enables the recovery of a greater number of intact nuclei with adequate results up to 24 h. (B) The inclusion of a wash step after antibody incubation does not visually appear to shift FANS populations. (C) Example of a failed FANS experiment with lack of distinct PAX6+(NeuN−) population (on right) despite the presence of intact nuclei and low background (on left) (postmortem temporal neocortex, non-pathological, 7 h PMI). (D) Heatmap representation of adult human sn RNA-seq data showing differential expression of PAX6 and two other astrocyte nuclear factors, SOX9 and LHX2, across protoplasmic and fibrous astrocyte subpopulations in both cortex and SVZ, compared to other cell types (red color gradient represents spectrum of log-normalized average gene expression values; n=3 distinct autopsy cases, 43,619 total nuclei. (E) Distinct brain regions (temporal neocortex vs. SVZ and subjacent striatum) show different patterns of NeuN/PAX6/OLIG2 separation. Abbreviations: FANS = fluorescence-activate nuclei sorting; DAPI = 4′,6-diamidino-2-phenylindole; NeuN = neuronal nuclei; PAX6 = paired box protein 6; OLIG2 = oligodendrocyte transcription factor 2; GFAP = glial fibrillary acidic protein; ALDH1L1 = 10-formyltetrahydrofolate dehydrogenase; LXH2 = LIM homeobox 2; PMI = postmortem interval; SSC-A = side scatter-area; FSC-A = forward scatter area; NeuN-555 = mouse anti-NeuN conjugated to AF555; PAX6-APC = mouse anti-PAX6 conjugated to allophycocyanin; OLIG2–488 = mouse anti-OLIG2 conjugated to green fluorescent dye for the 488 nm laser line; SVZ = subventricular zone; A(p) = protoplasmic (gray matter) astrocytes; A(f) = fibrous (white matter) astrocytes; OL = oligodendrocytes; OPC = oligodendrocyte progenitor cells; EN = excitatory neurons; MSN = medium spiny neurons; MG = microglia; BVC = blood vessel cells.