Abstract
Primary murine neurons are a well-established tool for investigating Tau in the context of neuronal development and neurodegeneration. However, culturing primary neurons is usually time-consuming and requires multiple feeding steps, media exchanges, proprietary media supplements, and/or preparation of complex media. Here, we describe (i) a relatively cheap and easy cell culture procedure for the cultivation of forebrain neurons from embryonic mice (E13.5) based on a commercially available neuronal supplement (NS21), (ii) a protocol for the cultivation of hippocampal and cortical neurons from postnatal (P0-P3) animals, and (iii) basic fixation and immunofluorescence techniques for the staining of neuronal markers and endogenous Tau. We demonstrate a staining technique, which minimizes antibody consumption and allows for fast and convenient processing of samples for immunofluorescence microscopy of endogenous Tau in primary neurons. We also provide a protocol that enables cryopreservation of fixed cells for years without measurable loss of Tau signal. In sum, we provide reliable protocols enabling microscopy-based studies of Tau in primary murine neurons.
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Acknowledgments
We thank Jennifer Klimek for her excellent technical support. Animals were provided by CMMC animal facility, CECAD in vivo research facility (both Cologne, Germany), and the Institute for Laboratory Animal Science, RWTH Aachen University, Faculty of Medicine, Aachen, Germany. This work was supported by the Koeln Fortune Program/Faculty of Medicine, University of Cologne, by the Else-Kröner-Fresenius-Stiftung, both to HZ, by a stipend from the Studienstiftung des deutschen Volkes to MBS, and by the “START-program” of the Faculty of Medicine of the RWTH Aachen University to NH.
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Buchholz, S., Bell-Simons, M., Haag, N., Zempel, H. (2024). Tracking Tau in Neurons: How to Grow, Fix, and Stain Primary Neurons for the Investigation of Tau in All Developmental Stages. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_29
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DOI: https://doi.org/10.1007/978-1-0716-3629-9_29
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