Pre-Embedding Immunostaining of Brain Tissue and Three-Dimensional Imaging with FIB-SEM

Turégano-López, Marta; Toledo-Rodriguez, Maria; Alonso‐Nanclares, Lidia; González‐Soriano, Juncal; DeFelipe, Javier; Merchán-Pérez, Ángel

doi:10.1007/978-1-0716-1522-5_20

Cited by 4 publications

(3 citation statements)

References 30 publications

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“…Focused ion beam milling and scanning electron microscopy serial sectioning can be combined with several labeling methods. These include genetic labeling ( Bosch et al, 2015 , 2016 ) tract-tracing ( Rodriguez-Moreno et al, 2018 , 2020 ) and immunocytochemistry ( Turégano-López et al, 2021 ). These methods allow the researcher to select a group of cells based on their origin, projections, or neurochemical characteristics.…”

Section: Discussionmentioning

confidence: 99%

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy

et al. 2022

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The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

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Section: Discussionmentioning

confidence: 99%

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy

et al. 2022

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“…When we wrote the first edition of Receptor and Ion Channel Detection in the Brain: Methods and Protocols, our aim was compiling and summarizing those commonly used techniques to study neurotransmitter receptors and/or ion channels. Now, in this second edition of the book, besides updating the original chapters, some of them substantially, we have included five new chapters (i.e.,Chapters 10,16,20,22,and 30) with cutting-edge techniques developed very recently. Importantly, while obtaining methodological information it is common to turn into several scattered sources, and sometimes protocols offer conflicting advice.…”

Section: Prefacementioning

confidence: 99%

Receptor and Ion Channel Detection in the Brain

2021

Neuromethods

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Experimental life sciences have two basic foundations: concepts and tools. The Neuromethods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of things as care has been taken to integrate these tools within the context of the concepts and questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to the methods and their development. Thus it gives the reader a better understanding of the origin of the techniques and their potential future development. The Neuromethods publishing program strikes a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques, including, for example, immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results.Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods series has been a success since its first volume published through Humana Press in 1985. The series continues to flourish through many changes over the years. It is now published under the umbrella of Springer Protocols. While methods involving brain research have changed a lot since the series started, the publishing environment and technology have changed even more radically. Neuromethods has the distinct layout and style of the Springer Protocols program, designed specifically for readability and ease of reference in a laboratory setting.The careful application of methods is potentially the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the relationships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly find sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy can therefore be acquired quickly and for a competitive price anywhere in the world.

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“…Subsequently, we used FIB-SEM to image cortical regions with VGAT-positive puncta, identifying synapses based on VGAT-positive boutons and unlabeled terminals. This material was prepared without potassium ferrocyanide, and the AS and SS were clearly identified and distinguished from one another (Turégano-López et al, 2021). However, volume electron microscopy studies have commonly employed potassium ferrocyanide Stevens, 1988, 1989;Harris et al, 1992Harris et al, , 2015Medalla et al, 2007;Hua et al, 2015) or potassium ferricyanide (Tapia et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

Cano-Astorga,

Plaza-Alonso,

Turegano-Lopez

et al. 2024

Front. Neuroanat.

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The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray’s type I and type II, corresponding to Colonnier’s asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

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Pre-Embedding Immunostaining of Brain Tissue and Three-Dimensional Imaging with FIB-SEM

Cited by 4 publications

References 30 publications

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy

Receptor and Ion Channel Detection in the Brain

Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

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