Os autores gostariam de agradecer Ikiel Ptito por seu contínuo apoio técnico.

Parte 1: Pré-processamento de tecido <ol><li> Tecido deve ser bem perfundidos com paraformaldeído, glutaraldeído ou formalina. Isto pode ser conseguido através de perfusão transcardial padrão normalmente utilizado para a colheita de outros órgãos. No presente estudo o assunto estava profundamente sedado com cloridrato de cetamina (10 mg / kg, im), sacrificados com uma overdose de pentobarbital sódico (25 mg / kg, iv) e perfundidos transcardially com 0,1 M PBS até que esteja completamente sangrar. Isso é seguido por uma solução de paraformaldeído 4% em PBS por 5 min (~ 1 litro). </li><li> O cérebro deve ser stereotaxically bloqueado, retirados do crânio, pesado, e volume determinado 2. O tecido deve então ser cryoprotected em soluções de sacarose classificados com uma concentração final de sacarose 30% em tampão fosfato. Blocos de tecido deve então ser congelados em isopentano a -65 ° C e armazenadas a -80 ° C até que esteja pronto para cortar. </li></ol> Parte 2: Amostragem Sistemática <ol><li> Amostragem sistemática depende de um bom plano antes do corte. Toda a região de interesse, tanto as extensões rostral-caudal e dorso-ventral devem ser bem definidos. Um atlas estereotáxico das espécies em uso é útil para definir a região de interesse e para determinar o comprimento da região de interesse. Uma vez que o comprimento da região de interesse é determinado, é necessário estabelecer o intervalo de amostragem. Por exemplo, desde o atlas estereotáxico-lo a determinar que a extensão rostro-caudal total da região de interesse é de aproximadamente 3 milímetros de comprimento quando o corte no plano coronal. Você, então, decidir o criostato a seção de 50 ìm, a este ritmo, terá 60 seções que cobrem a extensão rostral-caudal da região de interesse. Para um estudo imparcial típico estereológica 10/06 seções sistematicamente amostradas são necessários para obter resultados confiáveis. Neste exemplo, 10 seções igualmente espaçados ao longo do resultado extensão rostro-caudal em um intervalo de amostragem de 1 / 6 seções. </li></ol> Parte 3: Criação do banco de cérebros <ol><li> Uma vez que a frequência de amostragem foi determinada seção, o próximo passo é determinar quais as manchas serão realizados imediatamente. Por exemplo, se você estiver com coloração violeta cresil você irá capturar 06/01 seções em um slide. Esta será uma série. Se você sabe que há talvez vários anticorpos potencial que você gostaria de correr, mas tanto quer ver os dados que o seu principal fornece mancha ou simplesmente não têm o tempo para realizar a imuno-histoquímica, coloque o restante da série 5 seções na ordem em que eles foram cortados em poços preservar antígeno contendo (1 polivinil pirrolidona%, etileno glicol 50% em o.1M PBS, pH 7,4). A tabela abaixo fornece um sistema de amostragem composta por uma lâmina e uma placa (placa de 48 bem-padrão) de seções como parte de um banco de cérebros (Tabela 1). <img src="/files/ftp_upload/1260/table1.jpg" alt="Tabela 1" /> Tabela 1. A freqüência de amostragem seção aqui é 06/01. A primeira seção é colocado sobre uma lâmina para coloração violeta cresil e os próximos 5 seções são colocados em antígeno preservar. Número da seção 7 é então capturado no slide e seções 9-12 são colocados em antígeno preservar. Este ciclo é repetido até que a região de interesse ou bloco de tecido é exaustivamente seccionado. </li><li> O próximo passo é exaustivamente seção do bloco de tecido. Coloque uma pequena quantidade de incorporação de média no mandril e deixá-lo congelar. Coloque o chuck na cabeça micrótomo e raspar o suficiente do meio de congelados para ter uma superfície plana. Remova o mandril da cabeça do micrótomo e local plano dentro do criostato. Despeje sobre a incorporação de médio chuck e firmemente colocar o bloco do cérebro com o lado stereotaxically corte posicionado categoricamente sobre o mandril. Lentamente e completamente incorporar o cérebro no meio de montagem. Coloque o chuck com o cérebro na cabeça do micrótomo e seção usando os parâmetros pré-determinados para a frequência de amostragem seção. </li><li> Assim que o bloco de tecido tem sido exaustivamente seccionados e as secções são colocadas em poços, cobrir a placa com a tampa, envolva a tampa com parafilme, e colocar em um freezer -20 C °. Registrar o número total de seções tomadas para cada série, o intervalo de seção de amostragem, eo número de série para cada animal. Este log será vital para manter o controle de posterior remoção de seções do banco de cérebros para imunohistoquímica futuro. </li></ol> Parte 4: Resultados Representante: Amostragem sistemática desta maneira tem sido uma prática comum em nosso laboratório durante os últimos três anos. Tivemos um grande sucesso da imuno-histoquímica desempenho no material que foi armazenado em antígeno preservar três anos depois que foi cortada, sem deterioração do sinal (Figura 1). Além disso, como parte do nosso banco de cérebros, temos registrados cerca de 20.000 seções sistemática da não-humanosprimata cérebro (Figura 2) como parte de nossos planos de investigação a longo prazo. <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> Figura 1. Esta é uma seção do pólo occipital do primata não-humano histoquímica para NeuN mostrando tanto a camada VI e intersticial neurônios da substância branca. Esta seção especial foi armazenado em antígeno preservar a -20 ° C por um período de dois anos. Barra de escala = 100μm. <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="Figura 2" /> Figura 2. Nosso banco de cérebros vervet agora consiste de cerca de 20 mil seções sistemática de mais de 30 macacos.

<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.

Amostragem sistemática é um método barato pretende maximizar material de pesquisa. Esta estratégia de amostragem foi desenvolvido em conformidade com a legislação de estereologia imparcial que requer amostragem sistemática em toda a região. É fundamental que a ordem das secções para cada série é mantida. Em nosso laboratório temos utilizado com sucesso este método para operações bancárias tanto hamster e não-humanos seções cérebro dos primatas. Até agora, nós coletamos seções cérebro perto de ...

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brain banking: making the most of your research specimens

Estereologia imparcial é um método para estimar com precisão e eficiência o número de neurônios total (ou outro tipo de célula) em uma determinada área de interesse<sup&gt; 1</sup&gt;. Para atingir este objetivo 6-10 seções sistemática deve ser sondado cobrindo toda a estrutura. Normalmente, isso envolve o processamento 05/01 seções que deixa uma quantidade significativa de matérias não transformadas. A fim de maximizar o material, propomos um método barato para a preservação do tecido fixado no âmbito de um plano de pesquisa de longo prazo de armazenamento. Como o tecido é cortado e processado para a desejada mancha ou anticorpos, seções alternadas devem ser sistematicamente colocado em antígeno preservar a -20 ° C para processamento futuro. Usando placas de 24 poços, seções podem ser colocados em ordem para recuperação futura. Usando este método, o tecido pode ser armazenada e processada para imuno-histoquímica ao longo de anos.

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

Bancário cérebro e amostragem sistemática de material biológico fornece a base para estereologia imparcial e maximiza o potencial de dados obtidos de cada espécime.

Banking cérebro: Fazer oa maioria de espécimes sua Pesquisa

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 visualizações.  University of Montreal. Bancário cérebro e amostragem sistemática de material biológico fornece a base para estereologia imparcial e maximiza o potencial de dados obtidos de cada espécime.

Vídeo: 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-dissecção de cérebro de um rato para RNA ou Extração de Proteína da região específica do cérebro

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

Biologia

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.

Eletroporação única célula in vivo dentro do cérebro intacto desenvolvimento

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.

Tornando-Patch pipetas e Eletrodos Sharp com um extrator programável

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.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia 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.

Jogue Criança fazendo MR Imaging - Protocolo de neuroimagem Pediátrica, Diretrizes e Procedimento

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.

Fazendo Zebrafish Diplóide Gynogenetic por pressão 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.

Longo prazo Cultura de Amostras de mama Humano do Câncer e sua análise por meio de tomografia óptica de Projeção

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.

Obtenção de amostras com retardado, acelerado e invertida Envelhecimento no Modelo Bee Mel

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.

A amostragem de sangue a partir da veia lateral da cauda do rato

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: Um método de compensação por imagem tridimensional do tecido adiposo tecido

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

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Methods Article

Banking cérebro: Fazer oa maioria de espécimes sua Pesquisa