The protocol and reagents for the isolation of brain nuclei before the flow cytometry separation were described previously (10). In short, ~750 mg of tissue was homogenized in lysis buffer, underlaid with high sucrose buffer, and centrifuged for 1 hour at 24,000 rpm. The nuclear pellets were resuspended in the antibody-incubation buffer and incubated with primary antibodies for 1.5 hours. A second centrifugation step was performed, and nuclei were incubated with secondary antibodies for an additional 1 hour. A FANS method was then used to separate nuclei of MGE-GABA neurons, Glu neurons, and OLIG, similar to the protocol described in our previous publication (10), with some modifications. Antibodies against brain cell population markers NeuN, SOX6, and SOX10 were used in the FANS protocol. NeuN (also known as RNA-binding protein RBFOX3) is a well-established marker of neuronal nuclei (47). SOX6 is a transcription factor expressed in MGE-derived GABA neurons during development and into adulthood (48); the use of anti-SOX6 antibodies (1:1500, guinea pig polyclonal) (49) to separate the nuclei of MGE-derived GABA neurons from nuclei of Glu neurons was described by us previously (10). SOX10 is a transcription factor specifically expressed in OLIG. The application of anti-SOX10 antibodies (1:300, goat polyclonal; R&D Systems, AF2864) to isolate the nuclei of OLIG was described by Ernst and colleagues (20). For simultaneous FANS isolation of MGE-GABA, Glu, and OLIG nuclei, we used mouse monoclonal anti-NeuN phycoerythrin (PE)–conjugated antibodies (Millipore, FCMAB317PE, 1:1000), donkey anti-guinea pig AX647-conjugated secondary antibodies (Jackson ImmunoResearch, 1:1500) to detect SOX6-positive nuclei, and donkey anti-goat AX488-conjugated secondary antibodies (Life Technologies, A21447, 1:1500) to detect SOX10-positive nuclei. DNA stain 4′,6-diamidino-2-phenylindole was used to label intact nuclei.

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