Die Autoren bedanken sich bei Ikiel Ptito für seine weitere fachliche Unterstützung danken.

Teil 1: Pre-Processing von Gewebe <ol><li> Tissue sollte gut mit Paraformaldehyd, Glutaraldehyd oder Formalin perfundiert werden. Dies kann durch Standard-transcardial Perfusion in der Regel verwendet, um andere Organe Ernte erreicht werden. In der vorliegenden Studie das Thema war tief mit Ketaminhydrochlorid (10 mg / kg, im), mit einer Überdosis Natrium-Pentobarbital (25 mg / kg, iv) eingeschläfert sediert und perfundierten transkardial mit 0,1 M PBS, bis sie vollständig ausgeblutet. Dies wird durch eine 4% Paraformaldehyd-Lösung in PBS für 5 min (~ 1 Liter) folgten. </li><li> Das Gehirn soll stereotaxically blockiert, entfernt aus dem Schädel, gewogen und Volumen bestimmt 2. Das Gewebe sollte dann in abgestufter Saccharose-Lösungen mit einer Endkonzentration von 30% Saccharose in Phosphatpuffer cryoprotected werden. Blockiert von Gewebe sollte dann in Isopentan bei -65 ° C eingefroren und bei -80 ° C bis Sie bereit sind zu schneiden sind. </li></ol> Teil 2: Systematische Sampling <ol><li> Systematische Stichproben beruht auf einem richtigen Plan vor dem Schneiden. Die gesamte Region von Interesse sowohl in der rostral-caudalen und dorsalen-ventralen Ausmaß sollte genau definiert werden. Eine stereotaktische Atlas der Arten in Gebrauch ist hilfreich, um die Region von Interesse zu definieren und die Länge der Region von Interesse zu bestimmen. Sobald die Länge der Region von Interesse bestimmt ist es notwendig, das Abtastintervall zu etablieren. Zum Beispiel aus der stereotaktischen Atlas Sie feststellen, dass die gesamte rostro-kaudale Ausdehnung der Region von Interesse von ca. 3 mm in der Länge ist, wenn Schnitte in der Frontalebene. Sie entscheiden dann, mit dem Kryostaten Abschnitt gesetzt bei 50 um, in diesem Tempo werden Sie 60 Abschnitte für den rostral-kaudale Ausdehnung der Region von Interesse sind. Für eine typische unvoreingenommene stereologische Studie 6-10 systematisch abgetastet Abschnitte sind erforderlich, um zuverlässige Ergebnisse zu erhalten. In diesem Beispiel 10 Sektionen gleichmäßig über die rostro-kaudale Ausdehnung Ergebnis in einem Abtastintervall von 1 / 6 Abschnitte verteilt. </li></ol> Teil 3: Erstellung des Gehirns Bank <ol><li> Sobald der Abschnitt Abtastfrequenz bestimmt worden ist, ist der nächste Schritt, um zu bestimmen, die Flecken sofort durchgeführt werden. Zum Beispiel, wenn Sie mit Kresylviolett Färbung die Sie aufnehmen 1 / 6 Abschnitte auf einer Folie. Dies wird series 1 sein. Wenn Sie wissen, dass es vielleicht mehrere potenzielle Antikörper, die Sie gerne laufen, aber entweder wollen die Daten sehen, dass Ihre primäre Fleck stellt oder haben einfach nicht die Zeit, die Immunhistochemie durchzuführen, legen Sie die restlichen 5 Reihe von Abschnitten in der Reihenfolge ihres wurden in Vertiefungen mit Antigen zu erhalten (1% Polyvinylpyrrolidon, 50% Ethylenglykol in o.1M PBS, pH 7,4) in Scheiben geschnitten. Die folgende Tabelle bietet einen Stichprobenplan, bestehend aus 1 Rutsche und 1 Platte (Standard 48-Well-Platte) der Abschnitte als Teil eines Gehirns Bank (Tabelle 1). <img src="/files/ftp_upload/1260/table1.jpg" alt="Tabelle 1" /> Tabelle 1. Der Abschnitt Abtastfrequenz hier ist 1 / 6. Der erste Abschnitt ist auf einer Folie für Kresylviolett Färbung gelegt und die nächsten 5 Sektionen werden in Antigen platziert zu bewahren. Nummer des Abschnitts 7 wird dann auf der Folie erfasst und Abschnitte 9-12 sind in Antigen platziert zu bewahren. Dieser Zyklus wird wiederholt, bis die Region von Interesse oder die Sperrung von Gewebe ist erschöpfend geschnitten. </li><li> Der nächste Schritt ist, um abschließend Abschnitt des Blocks von Gewebe. Legen Sie eine kleine Menge Einbettmedium auf dem Futter und lassen Sie es einfrieren. Legen Sie das Futter in den Mikrotom Kopf und abrasieren genug von den gefrorenen Mediums auf eine ebene Oberfläche haben. Entfernen Sie das Futter aus dem Mikrotom Kopf und Stelle flach innerhalb des Kryostaten. Gießen Einbettmedium auf dem Futter und einen festen Platz im Gehirn blockieren mit dem stereotaxically Schnittseite flach auf dem Futter positioniert. Langsam und vollständig eingebettet in das Gehirn Eindeckmedium. Legen Sie das Futter mit dem Gehirn in das Mikrotom Kopf und Abschnitt mit dem vorher festgelegten Parameter für den Abschnitt Sampling-Frequenz. </li><li> Nachdem der Block des Gewebes wurde abschließend geschnitten und Abschnitte sind in Vertiefungen gegeben, decken Sie die Platte mit dem Deckel, wickeln Sie den Deckel mit Parafilm und in einem -20 ° C Gefrierschrank. Melden Sie sich die Gesamtzahl der Abschnitte für jede Serie, die section-Abtastintervall, und die Anzahl der Reihen für jedes Tier übernommen. Dieses Protokoll wird entscheidend sein, den Überblick über nachträgliche Entfernung von Teilen davon abhalten, das Gehirn Bank für zukünftige Immunhistochemie. </li></ol> Teil 4: Repräsentative Ergebnisse: Systematische Stichproben auf diese Weise eine gängige Praxis in unserem Labor in den letzten 3 Jahren. Wir hatten viel Erfolg Durchführung Immunhistochemie auf Material, das in Antigen gespeichert wurde erhalten drei Jahre nach, ohne eine Verschlechterung des Signals (Abbildung 1) in Scheiben geschnitten wurde. Darüber hinaus als Teil unseres Gehirns Bank haben wir rund 20.000 systematische Abschnitte des nicht-menschlichen angemeldetPrimatengehirn (Abbildung 2) als Teil unserer langfristigen Forschungs-Pläne. <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> Abbildung 1. Dies ist ein Ausschnitt aus dem okzipitalen Pol des nicht-menschlichen Primaten ist immungefärbt für NeuN zeigt sowohl Schicht VI und interstitielle weißen Substanz Nervenzellen. Dies insbesondere Abschnitt wurde in Antigen gespeichert bewahren bei -20 ° C für einen Zeitraum von 2 Jahren. Maßstab = 100 um. <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="figure2" /> Abbildung 2. Unsere vervet Gehirn Bank besteht heute aus rund 20.000 systematische Abschnitte aus über 30 Affen.

<ol>
	<li>West, M., Slomianka, L., Gundersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+the+subdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.+Aat+Rec.">Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Aat Rec.</a> 231, 482-497 (1991).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+Non-human+Primate+Brain+in+Stereotaxic+Space.">Dissecting the Non-human Primate Brain in Stereotaxic Space.</a> J Vis Exp. , (2009).</li></ol>

The authors have nothing to disclose.

Systematische Stichproben ist eine kostengünstige Methode soll Forschungsmaterial zu maximieren. Diese Strategie der Probenahme ist so konzipiert, mit dem geltenden nationalen unvoreingenommene Stereologie, dass die systematische Stichproben in der gesamten Region erfordert entsprechen. Es ist wichtig, dass die Reihenfolge der Abschnitte für jede Serie beibehalten wird. In unserem Labor haben wir erfolgreich diese Methode für Banken sowohl Hamster und nicht-menschlichen Primaten Hirnschnitten verwendet. Bisher haben ...

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<td>Ethylene glycol</td>
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brain banking: making the most of your research specimens

Unvoreingenommene Stereologie ist eine Methode zur genauen und effizienten Schätzung der insgesamt Neuron-Nummer (oder anderen Zelltyp) in einem bestimmten Bereich von Interesse<sup&gt; 1</sup&gt;. Um dieses Ziel zu erreichen 6-10 systematische Abschnitte sollten untersucht werden, die den gesamten Aufbau. Typischerweise geht mit der Verarbeitung 1 / 5 Abschnitte, die eine erhebliche Menge an Material unbearbeitet lässt. Um das Material zu maximieren, schlagen wir ein kostengünstiges Verfahren zur Konservierung von fixiertem Gewebe als Teil einer langfristigen Lagerung Forschungsplan. Als Gewebe wird in Scheiben geschnitten und für den gewünschten Fleck oder Antikörper, alternative Abschnitte verarbeitet werden, sollten systematisch in Antigen platziert werden erhalten bei -20 ° C für die zukünftige Verarbeitung. Mit 24-Well-Platten, können Abschnitte, um für den späteren Abruf gestellt werden. Mit dieser Methode kann Gewebe gespeichert und verarbeitet werden für die Immunhistochemie im Laufe der Jahre.

Watch this Scientific Journal Video about Brain Banking: Making the Most of your Research Specimens at JoVE.com

Brain Banking: Making the Most of your Research Specimens

Brain-Banking und systematische Entnahme von biologischem Material bildet die Grundlage für unvoreingenommene Stereologie und maximiert das Potenzial von Daten aus jeder Probe erhalten.

Brain-Banking: Making the Most Ihrer Forschung Proben

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

brain-banking-making-the-most-of-your-research-specimens

Research

JoVE Journal

Biology

9.9K Aufrufe.  University of Montreal. Brain-Banking und systematische Entnahme von biologischem Material bildet die Grundlage für unvoreingenommene Stereologie und maximiert das Potenzial von Daten aus jeder Probe erhalten.

Serie: Brain Banking: Making the Most of your Research Specimens

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-Dissektion Rattenhirns RNA oder Protein Extraction von bestimmten Hirnregion

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Forschung

Biologie

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single Cell Elektroporation in vivo innerhalb der intakten sich entwickelnde Gehirn

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Machen Patch-Pipetten und Sharp Elektroden mit einem programmierbaren Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Erstellen MR Imaging Kinderspiel - Pediatric Neuroimaging-Protokoll, Richtlinien und Verfahren

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Machen Gynogenetic Diploid Zebrafisch von Anfang Druck

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Langfristige Culture of Human Breast Cancer Proben und deren Analyse mit Hilfe von optischen Projektion Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Beziehen Proben mit verlangsamt, Beschleunigte und Umge Aging in der Honig-Bienen-Modell

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blutprobenentnahme aus der laterale Schwanzvene der Ratte

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...