We are grateful to Frank Ervin, Roberta Palmour and the staff of the Behavioural Sciences Foundation Laboratories located in St Kitts, West Indies, for their continued support of our primate work. This work was supported by a fellowship from Fragile X Research Foundation of Canada (FXRFC) to SZ and - operating grants from the Canadian Institutes of Health Research (AC) and the National Engineering and Research Council of Canada (MP).

Immunohistochemisty is one of the most widely used techniques for characterizing protein expression in the brain of various experimental animal models. It is relatively easy to conduct systematic immunohistochemical procedures on the brains of rodents and other common experimental models with similar brain size. However, there is no published work to our knowledge that provides a comprehensive account of how to carry out such immunodetection procedures across an entire monkey brain. What follows is a detailed description of how to prepare a whole monkey brain for large-scale immunohistochemical detection of various target proteins. This work has emerged as a result of collaboration between commercial and academic endeavours. As such, details pertaining to tissue embedding and sectioning remain a proprietary knowledge of NSA.
Part 1: Animal treatment and tissue preparation
The brain from an adult vervet monkey (Cercopithecus aethiops) is used for the present protocol. All procedures are carried out in compliance with the Canadian Council on Animal Care (CCAC) guidelines for the use and care of animals in biomedical research1.
<ol>
<li>Animal is deeply sedated with ketamine hydrochloride (10 mg/kg, i.m.), euthanized with an overdose of sodium pentobarbital (25 mg/kg, i.v.) and perfused transcardially with 0.1 M PBS until completely exsanguinated.</li>
<li>This is followed by a 4% paraformaldehyde solution in PBS for 5 min (~1 liter).</li>
<li>The externalized brain is placed in graded PBS-buffered sucrose solutions (10, 20 and 30%, in sequence) containing 0.02% sodium azide and maintained at 4&#x02DA;C until the brain sinks to the bottom of the container. The solution is replaced every few days until brain undergoes freeze-sectioning.</li>
<li>The brain is sent to NeuroScience Associates (NSA; Knoxville, TN) to be subjected to their proprietary MultiBrain&trade; embedding and freeze-sectioning technology.</li>
<li>Seven (7) alignment landmarks are placed in the embedding matrix so that sections can be oriented properly (i.e., left-right orientation) and re-aligned after histological processing is complete.</li>
<li>The frozen block is digitally photographed at the block face before each serial section is collected from the block. Digital images as well as sections from this brain can later be used for 3D reconstruction of anatomical and histological maps, respectively.</li>
<li>Serial free-floating coronal sections, at a thickness of 50 &micro;m per section, are collected from the entire brain.</li>
</ol>
Part 2: Histological processing
Sections are chosen at a given spatial interval (e.g., 500-&micro;m) and for our experimental purposes were processed with the following antibodies: fragile-X mental retardation protein (FMRP; Chemicon; Temecula, CA), SMI32 (Sternberger Monoclonals Inc.; Baltimore, MD), and NeuN (Chemicon; Temecula, CA). FMRP is a cytoplasmic protein that abundantly found in neurons of normal and permutation carrier brains2. NeuN (Neuronal Nuclei) specifically recognizes the DNA-binding neuron-specific protein NeuN which is present in most neurons and is distributed in neuronal nuclei, perikarya and some proximal neuronal processes3. SMI-32 is a monoclonal antibody that recognizes the non-phosphorylated epitope on neurofilament proteins4.
<ol>
<li>At an interval of 500 &micro;m, approximately 140 sections are used per antibody.</li>
<li>All incubations, washes and staining steps are carried out with mild agitation using specialized staining dishes and baskets (HistoTools; Knoxville, TN).</li>
<li>Sections are first incubated in a solution containing 0.1 M PBS/0.3% Triton X-100 (TX)/5% normal hoarse serum (NHS) for 60 min in order to reduce nonspecific binding of antibody molecules and resulting background staining.</li>
<li>Sections marked for FMRP immunostaining undergo an additional step of antigen recovery by using Antigen Unmasking Solution (Vector Labs; Burlingame, CA) according to vendor&rsquo;s instructions.</li>
<li>Sections are then incubated overnight and on separate days in their respective antibody solutions containing 0.1 M PBS/0.3% TX/3%NHS (with a dilution factor of 1/5,000 for FMRP and SMI-32 and 1/2,000 for NeuN antibodies.</li>
<li>The next day, sections are washed for three 10-min periods in washing solution (0.1 M PBS/0.3% TX) followed by incubation for 2 h at room temperature in biotinylated anti-mouse secondary antibody raised in horse (Vector Labs; 1/1,000 dilution in 0.1 M PBS/0.3% TX/3% NHS).</li>
<li>After a further set of 3 10-min washes, the sections are placed in a solution of avidin-biotin conjugated horseradish peroxidase complex (Vector Labs) for 1 h at room temperature.</li>
<li>Finally, after another set of washes, the sections are subjected for 10 min to a nickel enhanced diaminobenzidine (Ni-DAB) reaction which produces a dark blue stain within immunoreactive neurons.</li>
<li>At the completion of the histological procedure, sections are mounted on gelatin-coated glass slides.</li>
<li>All sections are air-dried overnight and coverslipped according to standard procedures.</li>
</ol>
Part 3: Representative Results:
This method produces a complete expression profile of a target protein of interest across an entire monkey brain. Here we show representative coronal sections that provide a snapshot of FMRP, NeuN and SMI32 expression in the same monkey brain.
<img src="/files/ftp_upload/1286/1286fig1.png" alt="" />
Figure 1: Staining dish (A) and basket (B) used for large-scale batch processing of free-floating sections for immunodetection.
<img src="/files/ftp_upload/1286/1286fig2.png" alt="" />
Figure 2: Representative coronal sections from a vervet monkey brain stained for FMRP (A), NeuN (B) and SMI32 (C) antibodies.

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

There are two critical steps in this procedure that make large-scale detection of proteins in an entire monkey brain possible. One is the embedding and sectioning protocol, which remains the proprietary knowledge of NSA. The other is the use of staining dishes and baskets provided by HistoTools. The latter allows for easy and quick handling of many (~40) sections at a given time. It also provides the means for uniformly treating all sections across the brain and makes for a scientifically sound histological treatment. In...

<table><tbody><tr><td>anti-FMRP monoclonal antibody</td><td>Chemicon International</td><td>MAB2160</td><td>Requires antigen retrieval in fixed tissue.</td></tr><tr><td>anti-NeuN monoclonal antibody</td><td>Chemicon International</td><td>MAB377</td><td>N/A</td></tr><tr><td>anti-Neurofilament H monoclonal antibody</td><td>Sternberger Monoclonals Inc.</td><td>SMI32</td><td>N/A</td></tr><tr><td>Antigen Unmasking Solution</td><td>Vector Laboratories</td><td>H-3300</td><td>N/A</td></tr><tr><td>Normal horse serum</td><td>Invitrogen</td><td>16050122</td><td>500 mL</td></tr><tr><td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td><td>Vector Laboratories</td><td>BA-2000</td><td></td></tr><tr><td>VECTASTAIN ABC Kit (Standard)</td><td>Vector Laboratories</td><td>PK-4000</td><td>1.5 mg</td></tr><tr><td>Staining dish for floating sections</td><td>HistoTools</td><td>10009</td><td>12 x 65 mm</td></tr><tr><td>Staining transport basket</td><td>HistoTools</td><td>10012</td><td>large&lt;br/&gt;(fits 12 x 65 mm dish)</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>P8514B</td><td>N/A</td></tr><tr><td>DAB</td><td>Fisher Scientific</td><td>AC11209-0050</td><td>N/A</td></tr></tbody></table>

batch immunostaining for large-scale protein detection in the whole monkey brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

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12.6K Views.  Montreal Neurological Institute. Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Video: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Neuroscience

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

Immunology and Infection

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Volumetric Imaging and Axonal Projection Mapping in the Marmoset Brain

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. We successfully implemented this technique for tracing the axonal projections in the marmoset brain. Here, the detailed experimental procedures that resulted in reliable volumetric imaging of the whole marmoset brain are described. A key process for successful imaging was the removal of meninges surrounding the brain, which interferes with slicing. A big advantage of this methodology is that the sliced sections can be used for additional staining. In the original set-up, the sliced sections are scrambled in the water bath. These sections can be correctly aligned in their original order according to the blood vessel patterns in the cortex. An example of effective histology is the visualization of myelin structure by simple light reflection, which can be combined with Nissl staining to define anatomical borders. These sections can also be used for immunological detection of non-fluorescent anterograde and retrograde tracers, which can be registered to the STPT data for layering of multiple data.

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic ...

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

<a href='https://app.jove.com/v/53853/combined-optogenetic-freeze-fracture-replica-immunolabeling-to'>Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)</a>This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate ...