Electroporated brains were dissected at the indicated embryonic ages, and successful electroporations were identified under an epifluorescence microscope. Brains with fluorescent labeling in the somatosensory cortex were fixed in a 4% formalin/PBS solution overnight at 4°C and cryoprotected in a 30% sucrose/PBS solution. Brains were frozen in optimal cutting temperature compound before 14-μm-thick coronal sections were obtained with a cryostat and placed on slides. Brain tissue was counterstained with DAPI before coverslipped with Fluoromount G mounting media. Most images were obtained with a Leica epifluorescent microscope on a 10× objective and captured with LAS X software. Images of three consecutive brain slices per brain were acquired.
To acquire high-magnification images, we used an Olympus Fluoview 3000 confocal laser scanning system on a 20× objective. Images were imported into Fiji for subsequent file conversion to TIFF format (RapID) or manual quantification (Cell Counter). For shCul5 results, raw images from a previously published study (Simó et al., 2010 (link)) were used.
To acquire high-magnification images, we used an Olympus Fluoview 3000 confocal laser scanning system on a 20× objective. Images were imported into Fiji for subsequent file conversion to TIFF format (RapID) or manual quantification (Cell Counter). For shCul5 results, raw images from a previously published study (Simó et al., 2010 (link)) were used.
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