Minimizing shrinkage of acute brain slices using metal spacers during histological embedding
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Minimizing shrinkage of acute brain slices using metal spacers during histological embedding Felix Bolduan1 · Sabine Grosser1 · Imre Vida1 Received: 9 April 2020 / Accepted: 2 September 2020 © The Author(s) 2020
Abstract The morphological structure of neurons provides the basis for their functions and is a major focus of contemporary neuroscience studies. Intracellular staining of single cells in acute slices is a well-established approach, offering high-resolution information on neuronal morphology, complementing their physiology. Despite major technical advances, however, a common histological artifact often precludes precise morphological analysis: shrinkage of the sampled tissue after embedding for microscopy. Here, we describe a new approach using a metal spacer, sandwiched between two coverslips to reduce shrinkage of whole-mount slice preparations during embedding with aqueous mounting medium under a coverslip. This approach additionally allows imaging the slices from both sides to obtain better quality images of deeper structures. We demonstrate that the use of this spacer system can efficiently and stably reduce the shrinkage of slices, whereas conventional embedding methods without spacer or with agar spacer cause severe, progressive shrinkage after embedding. We further show that the shrinkage of slices is not uniform and artifacts in morphology and anatomical parameters produced cannot be compensated using linear correction algorithms. Our study, thus, emphasizes the importance of preventing the deformation of slice preparations and offers an effective means for reducing shrinkage and associated artifacts during embedding. Keywords Acute brain slice · Single cell morphology · Biocytin labeling · Histology · Tissue shrinkage · Confocal imaging
Introduction The morphology of neurons provides the structural framework for their functions, including the integration of synaptic inputs and the generation of action potentials (Kasper et al. 1994; Norenberg et al. 2010; DeFelipe et al. 2013; de Sousa et al. 2015; Gulledge and Bravo, 2016; Mihaljevic et al. 2018). Indeed, from Cajal’s morphological studies of Golgi stained neurons (Ramón y Cajal 1909, 1911) up to today’s high-resolution confocal images of genetically identified and biocytin stained neurons (Bartos et al. 2002; Thomson and Armstrong 2011; Booker et al. 2014), anatomical investigations offered important insights for the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00429-020-02141-3) contains supplementary material, which is available to authorized users. * Imre Vida [email protected] 1
Institute of Integrative Neuroanatomy, Charité Universitätsmedizin Berlin, Berlin, Germany
understanding of the physiological and circuit functions of neurons and also provided essential data for computational analysis (Traub et al. 1994; Major et al. 1994; Norenberg et al. 2010; Gidon et al. 2020). While identification, labeling and imaging of neurons have shown substantial advances in
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