著者は、彼の継続的な技術サポートのためにIkiel Ptitoに感謝します。

パート1：組織の前処理<ol><li>組織はよくパラホルムアルデヒド、グルタルアルデヒド、またはホルマリンで灌流してください。これは通常、他の臓器を収穫するために使用される標準transcardial灌流によって達成することができます。本研究では被験者は深く、（10 mg / kgを、IM）塩酸ケタミンで鎮静したペントバルビタールナトリウムの過剰投与（25 mg / kgを、IV）を使用して、安楽死させ、完全に放血するまで0.1MのPBSでtranscardially灌流。これは、5分（〜1リットル）のためのPBS中4％パラホルムアルデヒド溶液が続いている。 </li><li>脳は、定位、ブロック頭蓋骨から削除、秤量し、ボリュームは2を決定する必要があります。組織は、その後リン酸緩衝液で30％のショ糖の最終濃度に段階的スクロース溶液で凍結保護されるべきである。組織のブロックがその後-65 ° Cでイソペンタンで凍結し、-80で保存してください° C、スライスする準備が整うまで。 </li></ol>パート2：システマティックサンプリング<ol><li>系統的サンプリングは、前の切片に適切な計画に依存しています。吻側 - 尾と背腹のエクステントの両方に関心の全領域は明確に定義されるべきである。使用中の種の脳地図は、関心領域を定義し、関心領域の長さを決定しておくと便利です。かつて関心領域の長さは、それがサンプリング間隔を確立する必要がある決定されます。例えば、脳地図からあなたは、冠状面でのセクショニング時に関心領域の全体rostro -尾程度の長さが約3mmであると判断する。次に、50μmの時にセクションにクライオスタットを設定するには、このレートでは関心領域の吻側 - 尾程度をカバーする60のセクションを持って決める。典型的な立体的研究のために60から10まで体系的にサンプリングされたセクションは、信頼できる結果を得るために必要とされています。この例では10のセクションでは、均等に6分の1のセクションのサンプリング間隔でrostro -尾程度の結果全体に等間隔。 </li></ol>パート3：脳バンクの作成<ol><li>節のサンプリング周波数が決定されると、次のステップは汚れがすぐに実行されるかを決定することです。たとえば、クレシルバイオレットで染色している​​場合には、スライドの1 / 6つのセクションをキャプチャします。これはシリーズ1になります。あなたはそこにあなたが実行したいが、どちらをプライマリ染み提供するデータを参照するか、単に免疫組織化学を実行する時間を持っていない、彼らはその順序でセクションの残りの5シリーズを配置することかもしれないいくつかの潜在的な抗体を知っていれば抗原保護区域 （1％ポリビニルピロリドン、o.1M PBS、pH7.4中50％エチレングリコール）を含むウェルにスライスされた。下の表は、1スライドと脳バンク（表1）の一環として、セクションの1枚（標準48ウェルプレート）で構成されるサンプリング方式を提供します。 <img src="/files/ftp_upload/1260/table1.jpg" alt="表1" /> 表1ここにセクションのサンプリング周波数は1 / 6です。最初のセクションは、クレシルバイオレット染色用スライド上に配置され、次の5つのセクションが保持する抗原に配置されます。セクションの番号7は、スライド上に捕獲され、セクションは、9月12日維持抗原に配置されます。組織の利益またはブロックの領域が網羅的に区分されるまで、このサイクルが繰り返されます。 </li><li>次のステップは、徹底的にセクションに組織のブロックです。チャックでメディアを埋め込むの少量を置き、それがフリーズしましょう​​。ミクロトーム頭にチャックを配置し、平坦な表面を持つように凍結培地を十分に剃り落とす。ミクロトームヘッドとクライオスタット内で平坦な場所からチャックを削除します。チャックでメディアを埋め込む注ぎ、しっかりとチャックを平らに置か定位カット側で脳のブロックを設置。ゆっくりと完全に培地をマウントするに脳を埋め込みます。ミクロトームヘッドと節のサンプリング周波数のためにあらかじめ決められたパラメータを使用してセクションに脳でチャックを置きます。 </li><li>組織のブロックが徹底的に区分されていると節をウェルに配置されると、、蓋でカバープレートをパラフィルムで蓋をラップし、-20℃の冷凍庫内の場所。各シリーズ、セクション、サンプリング間隔、およびそれぞれの動物のための一連の数に要したセクションの合計数をログに記録します。このログは、将来の免疫組織化学のために脳の銀行からセクションのその後の除去を追跡することが重要になります。 </li></ol>パート4：代表的な結果： この方法で体系的なサンプリングは、過去3年間、私たちの研究室で標準的となっています。我々は、それが信号の劣化（図1）することなくスライスした後三年を維持する抗原に格納されている材料に関する免疫組織化学を行う大きな成功があった。さらに、私たちの脳の銀行の一部として我々は非人間の近い〜20,000体系的セクションを記録している霊長類の脳（図2）当社の長期的な研究計画の一環として。 <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> 図1。NeuNは層VIと間質性白質のニューロンの両方を表示するための免疫染色これは、非ヒト霊長類の後頭極からセクションです。この特定のセクションは、2年間の期間は-20℃で保存する抗原に格納されていました。スケールバー=100μmの。 <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="図2" /> 図2我 々のベルベット脳の銀行は現在30以上のサルから近いから20,000の体系のセクションで構成されています。

<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.

系統的サンプリングは、研究材料を最大にするため安価な方法です。このサンプリングの戦略は、地域全体の体系的なサンプリングを必要とする公平な立体学の規則を遵守するように設計されています。それは、シリーズごとのセクションの順序が維持されていることが重要です。私たちのラボでは、正常ハムスターとヒト以外の霊長類の脳の両方のセクションをバンキングのこのメソッド?...

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brain banking: making the most of your research specimens

公平な立体学が正確かつ効率的に関心のある一定の領域で合計ニューロンの数を（または他の細胞型）を推定するための方法です。<sup&gt; 1</sup&gt;。 60から10まで体系的なセクションを、この目標を達成するため、構造全体をカバーするプローブする必要があります。通常、これは未処理材のかなりの量を残す5分の1のセクションの処理を伴います。素材を最大にするために、我々は長期保存の研究計画の一環として、固定した組織を維持するための安価な方法を提案する。組織が必要な汚れや抗体、別のセクションのためにスライスして処理される際には、体系的に将来の処理のために-20℃で保存する抗原に配置する必要があります。 24ウェルプレートを使用して、セクションは、将来の検索のための順序で配置することができます。この方法を使用して、組織が保存され、年間の経過、免疫組織化学のために処理することができます。

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

脳の銀行と生体物質の系統的サンプリングは、公平な立体学の基礎を提供し、各試料から得られる潜在的なデータを最大限に高めることができます。

脳バンキング：あなたの研究の標本のほとんどを作る

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 ビュー.  University of Montreal. 脳の銀行と生体物質の系統的サンプリングは、公平な立体学の基礎を提供し、各試料から得られる潜在的なデータを最大限に高めることができます。

ビデオ: 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.

RNAまたは特定の脳領域からのタンパク質抽出用のラット脳の微小解剖

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

生物学

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.

無傷の開発脳内の生体内の単一細胞のエレクトロポレーション

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.

パッチピペットおよびプログラマブルプラーとシャープ電極作成

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.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

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.

小児神経イメージングプロトコル、ガイドラインと手順 - MRイメージングチャイルドプレイを作る

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.

初期の圧力による雌性発生二倍体のゼブラフィッシュを作成

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.

光学投影トモグラフィを用いたヒト乳癌検体とその分析の長期培養

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.

ハニービーモデルの老化、減速し加速し、逆に試験片を得た

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.

ラットの外側尾静脈から血液をサンプリング

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.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

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