A confocal Z-stack of neurons in postnatal hippocampus, expressing a GFP-tagged knockdown construct for the microRNA miR-137 (green, sh-miR-137) and immunostained to localize doublecortin (red), a marker of neuronal differentiation, and labeled with Dapi (blue), to determine the function of this microRNA in vivo. miR-137 regulates a core transcription factor in stem cell renewal, and data from these experiments suggest that high levels of miR-137promotes neuronal stem cell proliferation, but repressed neuronal differentiation. This image is original data from Fig. 5 of Szulwach et al.(2010) Cross talk between microRNA and epigenetic regulation in adult neurogenesis, J. Cell Biol. Vol. 189:127–141.
A retroviral vector containing sh-miR-137 under a U6 promoter and EGFP under a chicken actin promoter was prepared, and the virus containing the construct was injected into the dentate gyrus of adult mice in vivo. After 1 week, animals were anesthetized, perfused with 4% paraformaldehyde, brains removed and postfixed, equilibrated in 30% sucrose, and sectioned at a thickness of 40µm with a sliding microtome. Infected cells were localized using chicken-α-GFP (Invitrogen) and rabbit-α-DCX (Cell Signaling Technology), secondaries were α-chicken Alexa Fluor 488 and goat-α-rabbit Alexa Fluor 568 (both from Invitrogen). Confocal z-stacks were acquired at 1 µm steps using a Nikon TE2000 with equipped with a spinning disc confocal with an oil immersion 40X 1.3 NA objective (Zeiss). For additional details see: J. Cell Biol. Vol. 189:127–141.
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