Los autores desean agradecer a Ikiel Ptito por su apoyo técnico continuo.

Parte 1: Pre-procesamiento de tejidos <ol><li> Tejido debe ser buena perfusión con paraformaldehído, glutaraldehído, o formalina. Esto se puede lograr a través de la perfusión estándar transcardial normalmente se utiliza para la cosecha de otros órganos. En el presente estudio el tema fue profundamente sedado con clorhidrato de ketamina (10 mg / kg, im), sacrificados con una sobredosis de pentobarbital sódico (25 mg / kg, iv) y perfundidos transcardially con PBS 0,1 M hasta que esté completamente desangrado. Esto es seguido por una solución de paraformaldehído al 4% en PBS durante 5 minutos (aproximadamente 1 litro). </li><li> El cerebro debe ser stereotaxically bloqueado, removido del cráneo, pesado y determinado volumen 2. El tejido debe ser crioprotegieron en soluciones de sacarosa calificado con una concentración final de 30% de sacarosa en tampón fosfato. Bloques de tejido deberá congelarse en isopentano a -65 º C y almacenadas a -80 ° C hasta que esté listo para cortar. </li></ol> Parte 2: Muestreo sistemático <ol><li> El muestreo sistemático se basa en un plan adecuado antes de cortar. Toda la región de interés, tanto en la extensión rostral-caudal y dorsal-ventral deben estar bien definidos. Un atlas estereotáxico de la especie en uso es útil para definir la región de interés y para determinar la longitud de la región de interés. Una vez que la longitud de la región de interés se determina que es necesario establecer el intervalo de muestreo. Por ejemplo, desde el atlas estereotáxico a determinar que todo el rostro-caudal extensión de la región de interés es de aproximadamente 3 mm de longitud cuando se corte en el plano coronal. A continuación, decide establecer el criostato a la sección a 50 micras, a este paso, tendrá 60 secciones que abarcan la extensión rostral-caudal de la región de interés. Para un típico estudio imparcial estereológicos 10.06 secciones muestreados de manera sistemática se requiere para obtener resultados fiables. En este ejemplo, 10 secciones igualmente espaciados a lo largo de la medida resultado rostro-caudal en un intervalo de muestreo de 1 / 6 secciones. </li></ol> Parte 3: Creación de un banco de cerebros <ol><li> Una vez que la frecuencia de muestreo de la sección se ha determinado, el siguiente paso es determinar que las manchas se llevará a cabo de inmediato. Por ejemplo, si usted es la tinción con violeta de cresilo que captará 6.1 secciones en una diapositiva. Esta será la serie 1. Si usted sabe que hay quizá varias posibles anticuerpos que le gustaría correr, pero, o bien quieren ver los datos que proporciona la principal mancha o simplemente no tienen el tiempo para llevar a cabo la inmunohistoquímica, coloque el restante 5 series de secciones en el orden en que se cortan en preservar los pozos que contienen antígeno (1 polivinilpirrolidona%, 50% de glicol de etileno en o.1M PBS, pH 7,4). La siguiente tabla muestra un esquema de muestreo que consta de un tobogán y una placa (placa estándar de 48 pocillos) de las secciones como parte de un banco de cerebros (Tabla 1). <img src="/files/ftp_upload/1260/table1.jpg" alt="Tabla 1" /> Tabla 1. La frecuencia de muestreo sección aquí es 1 / 6. La primera sección se coloca en un portaobjetos para la tinción de violeta de cresilo y los próximos cinco secciones se colocan en el antígeno preservar. Número de la sección 7 es capturado en la diapositiva y las secciones se colocan en 9.12 antígeno preservar. Este ciclo se repite hasta la región de interés o un bloque de tejido se agota en secciones. </li><li> El siguiente paso es exhaustiva sección del bloque de tejido. Coloque una pequeña cantidad de medio de inclusión en el plato y deje que se congele. Coloque el plato en la cabeza del microtomo y afeitarse suficiente del medio congelados para tener una superficie plana. Quitar el plato de la cabeza microtomo y lugar plano en el criostato. Verter medio de inclusión en el plato y colocar el bloque con firmeza el cerebro con la parte cortada stereotaxically posicionado rotundamente en el plato. Lenta y completamente integrar el cerebro en medio de montaje. Coloque el plato con el cerebro en la cabeza del microtomo y la sección con los parámetros predeterminados para la frecuencia de muestreo de la sección. </li><li> Una vez que el bloque de tejido ha sido exhaustivamente seccionado y las secciones se colocan en los pozos, cubrir la placa con la tapa, ponga la tapa con parafilm, y colóquelo en un congelador a -20 ° C. Registrar el número total de secciones tomadas de cada serie, el intervalo de la sección de muestreo, y el número de serie de cada animal. Este registro será de vital importancia para realizar un seguimiento de la posterior retirada de las secciones del banco de cerebros de la inmunohistoquímica en el futuro. </li></ol> Parte 4: Resultados del representante: Muestreo sistemático de esta manera ha sido una práctica habitual en nuestro laboratorio durante los últimos 3 años. Hemos tenido un gran éxito la realización de inmunohistoquímica en el material que ha sido almacenado en el antígeno de preservar tres años después de que fue cortado, sin deterioro de la señal (Figura 1). Además, como parte de nuestro banco de cerebros que han registrado cerca de 20.000 secciones sistemática de los no humanoscerebro de los primates (Figura 2) como parte de nuestros planes de investigación a largo plazo. <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> Figura 1. Esta es una sección del polo occipital de los primates no humanos immunostained para NeuN que muestra tanto la capa VI e intersticiales neuronas de sustancia blanca. Esta sección en particular se almacena en el antígeno conservar a -20 ° C por un período de 2 años. Barra de escala = 100μm. <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="figura 2" /> Figura 2. Nuestro banco de cerebros de los monos verdes se compone actualmente de cerca de 20.000 secciones sistemática de más de 30 monos.

<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.

El muestreo sistemático es un método barato destinado a maximizar el material de investigación. Esta estrategia de muestreo se ha diseñado para cumplir con las normas de estereología imparcial que requiere un muestreo sistemático en toda la región. Es muy importante que el orden de las secciones de cada serie se mantiene. En nuestro laboratorio hemos utilizado con éxito este método para la banca de los hamster y no humanos, cortes de cerebro de los primates. Hasta ahora, hemos recogido cerca de 20.000 monos ver...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ethylene glycol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E178-4</td>
<td>&nbsp;</td>
<tr>
<td>Polyvinyl pyrrolidone</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP431-500</td>
<td>&nbsp;</td>
<tr>
<td>Thermal Scientific Embedding Medium</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

brain banking: making the most of your research specimens

Estereología imparcial es un método de forma precisa y eficiente la estimación del número de neuronas total (o otro tipo de células) en un área de interés<sup&gt; 1</sup&gt;. Para lograr este objetivo 60-10 secciones sistemática debe ser probado que cubre toda la estructura. Generalmente, esto implica el procesamiento 1.5 secciones que deja una importante cantidad de material sin procesar. Con el fin de maximizar el material, se propone un método barato para la preservación de tejido fijado como parte de un plan de almacenamiento a largo plazo de la investigación. Medida que el tejido se corta y se procesaron para la deseada mancha o anticuerpos, las secciones alternativas deberían ser sistemáticamente colocado en el antígeno conservar a -20 ° C para el procesamiento futuro. Utilizando placas de 24 pocillos, las secciones se pueden colocar en orden para su futura recuperación. Usando este método, el tejido puede ser almacenada y procesada por inmunohistoquímica en el transcurso de los años.

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

Banca cerebro y el muestreo sistemático de la materia biológica constituye la base para estereología imparcial y maximiza el potencial de los datos obtenidos de cada ejemplar.

Banca cerebro: Aprovechar al máximo las muestras de su investigación

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 vistas.  University of Montreal. Banca cerebro y el muestreo sistemático de la materia biológica constituye la base para estereología imparcial y maximiza el potencial de los datos obtenidos de cada ejemplar.

Video: 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-disección de cerebro de rata para el ARN o la extracción de proteínas a partir de región específica del cerebro

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

Investigación

Biología

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.

La electroporación in vivo sola célula en el cerebro intacto desarrollo

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.

Hacer patch pipetas y electrodos de Sharp con un extractor programable

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.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

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.

Hacer jugar MR Imaging Niño - Protocolo neuroimagen pediátrica, Directrices y Procedimientos

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.

Hacer pez cebra Gynogenetic diploides por presión inicial

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.

A largo plazo la cultura de muestras humanas del cáncer de pecho y su análisis mediante tomografía óptica de proyección

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.

La obtención de especímenes con desacelerado, acelerado y Invertida Envejecimiento en la abeja de la miel Modelo

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.

Toma de muestras de sangre de la vena lateral de la cola de la rata

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-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

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