Increasing numbers of studies seek to characterize the different cellular sub-populations present in mammalian tissues. The techniques “Isolation of Nuclei Tagged in Specific Cell Types” (INTACT) or “Fluorescence-Activated Nuclei Sorting” (FANS) are frequently used for isolating nuclei of specific cellular subtypes. These nuclei are then used for molecular characterization of the cellular sub-populations. Despite the increasing popularity of both techniques, little is known about their isolation efficiency, advantages, and disadvantages or downstream molecular effects. In our study, we compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 × CAG-Sun1/sfGFP animals. We identified differences in efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states. Therefore, our study presents a comprehensive comparison between the two widely used nuclei sorting techniques, identifying the advantages and disadvantages for both INTACT and FANS. Our conclusions are summarized in a table to guide researchers in selecting the most suitable methodology for their individual experimental design.

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The mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells is still a biochemically complex process, with every step affecting downstream results. Therefore, it is necessary to use protocols that fit the experimental design to yield nuclei of high quality and quantity. However, the current protocols are not suitable for nuclei isolation of small volumes of micro-dissected brain regions from individual mouse brains.Additionally, the caveats associated with centrifugation steps of nuclei extraction and the effects of different buffers have not been thoroughly investigated. Therefore, in this study, we describe an iodixanol based density gradient ultracentrifugation protocol suitable for micro-dissected brain regions from individual mice using ArccreERT2 (TG/WT).R26CAG-Sun1-sfGFP-Myc (M/WT or M/M). This mouse model shows sfGFP expression (sfGFP+) in the nuclear membrane of specific stimulus activated cells, thereby providing a good basis for the study - nuclei isolation and separation of cell-type-specific nuclei. The study also introduces new tools for rapid visualization and assessment of quality and quantity of nascent extracted nuclei. These tools were then used to examine critical morphological features of nuclei derived from different centrifugation methods and the use of different buffers to uncover underlying effects. Finally, to obtain cell-type-specific nuclei (sfGFP+ nuclei) from the isolated nuclei pool of high viscosity, an optimized protocol for fluorescence activated nuclei sorting (FANS) was established to speed up sorting. Additionally, we present a 1% PFA protocol for fixation of isolated nuclei for long term microscopic visualization.

Alzheimer's disease (AD) pathology precedes the onset of clinical symptoms by several decades. Thus, biomarkers are required to identify prodromal disease stages to allow for the early and effective treatment. The methoxy-X04-derivative BSC4090 is a fluorescent ligand which was designed to target neurofibrillary tangles in AD. BSC4090 staining was previously detected in post-mortem brains and olfactory mucosa derived from AD patients. We tested BSC4090 as a potential diagnostic marker of prodromal and early AD using olfactory mucosa biopsies from 12 individuals with AD, 13 with mild cognitive impairment (MCI), and 10 cognitively normal (CN) controls. Receiver-operating curve analysis revealed areas under the curve of 0.78 for AD versus CN and of 0.86 for MCI due to AD versus MCI of other causes. BSC4090 labeling correlated significantly with cerebrospinal fluid levels of tau protein phosphorylated at T181. Using NMR spectroscopy, we find that BSC4090 binds to fibrillar and pre-fibrillar but not to monomeric tau. Thus, BSC4090 may be an interesting candidate to detect AD at the early disease stages.