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Immunofluorescence staining of neuronal cultures
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Primary cultures of dissociated hippocampal neurons were fixed at 3 days in vitro (d.i.v.) with 4% paraformaldehyde/15% sucrose in phosphate buffered saline (PBS) for 20 min, permeabilized with 0.01% Triton X-100/0.1% Na-Citrate/PBS for 10 min on ice and stained with primary and secondary antibodies in 10% NGS/PBS. A Zeiss LSM 700 or LSM 800 confocal laser scanning microscope was used for imaging. Image analysis was done using ImageJ 1.45 s (NIH), and Adobe Photoshop CS5. The stage of neuronal differentiation was determined as described previously5.
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