Chunk #34 — ONLINE METHODS — Generating and Characterizing Isogenic ApoE3/3 hiPSC Lines by Zinc Finger Nuclease (ZFN)–mediated Gene Editing

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Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector.

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For immunostaining, hiPSCs cultured in 24-well plates were fixed with 4% paraformaldehyde and stained with primary antibodies against Nanog (ab21624, Abcam), human nuclei (MAB1281, EMD Millipore), Sox2 (sc-17320, Santa Cruz Biotechnology), Oct-3/4(sc-5279, Santa Cruz Biotechnology), SSEA4 (ab16287, Abcam), TRA-1-81 (MAB4381, EMD Millipore), and TRA-1-60 (MAB4360, EMD Millipore). The secondary antibodies were IgG conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Life Technologies). Nuclei were stained with DAPI. Images were taken with a Leica epifluorescence microscope, a Keyence BZ-9000E fluorescence microscope, or a Leica confocal imaging system.