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Fig. 6 | Fluids and Barriers of the CNS

Fig. 6

From: Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

Fig. 6

Overview of the main steps for processing a CNS tissue sample into BBB-related material ready for RNA isolation. Fresh samples are dissociated by mechanical disruption, enzymatic digestion, or a combination of both. Typically, tissue is first mechanically disaggregated into smaller pieces to facilitate the exposure to the enzyme solution. Dissociated tissue is then selected according to size by passing through one or a series of filters, by a density gradient, or both. This process isolates the microvessels. For isolating single barrier cells, tissue dissociation (particularly enzymatic digestion) can be repeated [1] after the initial size selection steps. The single cell suspension can be further purified or enriched for certain cell types [2] by using a fluorescence-activated cell sorter (FACS) or magnetic microbeads labeled with an antibody against a cell marker. Alternatively, if the tissue of interest is frozen or formalin-fixed paraffin-embedded (FFPE), a common approach is to isolate the microvessels by laser capture microdissection (LCM)

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