Abstract
FIB-SEM is an electron microscopy technique that allows the acquisition of serial sections in an automated manner. A Focused Ion Beam (FIB) is directed toward the specimen, removing material from its surface. Since the FIB can be positioned and controlled on a nanometer scale, the specimen surface can be milled so that a thin layer of a specified thickness is removed. The Scanning Electron Microscope (SEM) column is then used to acquire an image from the freshly milled surface. To obtain a series of images, the milling/imaging cycle is repeated automatically. This technique can be used with any pre-embedding method that yields an electron-dense end product. We describe here an immunocytochemical protocol to be used on vibratome brain sections. After the immunocytochemical procedure, the sections are flat embedded in epoxy resin and prepared for FIB-SEM imaging. Serial image acquisition, visualization, and analysis in 3D are also briefly described.
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Acknowledgments
The authors would like to thank L. Valdés and M.C. Álvarez for their technical assistance and Nick Guthrie for his excellent text editing. This work was partially supported by grants from the following entities: Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain); and the Spanish Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]).
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Turégano-López, M., Rodríguez, JR., Alonso-Nanclares, L., González-Soriano, J., DeFelipe, J., Merchán-Pérez, A. (2021). Pre-Embedding Immunostaining of Brain Tissue and Three-Dimensional Imaging with FIB-SEM. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_20
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DOI: https://doi.org/10.1007/978-1-0716-1522-5_20
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