作者想感谢他持续的技术支持Ikiel Ptito。

第1部分：前处理的组织<ol><li>组织应多聚甲醛，戊二醛，或福尔马林灌注。这可以通过标准transcardial，通常用于收获其他器官灌注。在本研究的主题是深刻的盐酸氯胺酮（10毫克/公斤，IM）与戊巴比妥钠（25毫克/公斤，四）过量安乐死，镇静剂和用0.1 M PBS灌注，直到完全exsanguinated transcardially人。其次是在PBS的4％多聚甲醛为5分钟（约1升）的解决方案。 </li><li>大脑应stereotaxically封锁，从头骨取出，称重，量确定2。组织，然后应cryoprotected分级蔗糖溶液与30％蔗糖磷酸盐缓冲液中的终浓度。组织块应被冻结在戊烷在-65 ° C，并保存于-80 ° C，直到您准备切片。 </li></ol>第2部分：系统抽样<ol><li>系统抽样依赖于适当的计划，以切片前。应该很好地确定整个地区的喙尾鳍和背腹程度的兴趣。一个使用物种的立体地图是有帮助的定义感兴趣的区域，并确定感兴趣的区域的长度。一旦感兴趣的区域的长度是确定有必要建立的采样间隔。例如，从立体地图集您确定整个rostro尾鳍的感兴趣的区域范围是大约3mm的长度时，在冠状面切片。然后，您决定设置的低温恒温器第50微米，在这个速度，你将有60节，包括感兴趣区域的喙尾鳍程度。 6-10系统采样的部分，对于一个典型的无偏体视学研究需要获得可靠的结果。在这个例子中10个章节等距整个采样间隔的1 / 6节rostro尾程度的结果。 </li></ol>第3部分：大脑银行的创建<ol><li>一旦部分采样频率已经确定，下一步是要确定哪些污渍，将立即执行。例如，如果你是用甲酚紫染色，你会捕捉幻灯片上的1 / 6节。这将是系列的第1。如果你知道有可能一些潜在的抗体，你想运行，但，他们是希望看到的数据，您的主要污渍提供或根本没有时间进行免疫组化，放置的顺序，其余5节系列切成井含有抗原的保护（1％聚乙烯吡咯烷酮，乙二醇50％，在o.1M PBS，pH值7.4 ）。下面的表格提供了一个抽样方案，包括1张幻灯片和1盘为大脑银行（见表1）部分路段（标准48孔板）。 <img src="/files/ftp_upload/1260/table1.jpg" alt="表1" /> 表1：这里的部分采样频率的1 / 6 。第一部分是放置甲酚紫染色的幻灯片和未来5节到抗原放置保存。幻灯片上的第7号，然后捕获和部分9-12放置在抗原保留。这种循环反复，直到该地区的利益或组织块详尽的切片。 </li><li>下一步是要详尽的部分组织块。将少量的嵌入夹头中等，让它冻结。放入切片头夹头和刮胡子的冷冻介质，足有一个平坦的表面。从切片机头部和地方单位的低温恒温器内取出夹头。倒入嵌入夹头介质和牢固的地方定位夹头断然stereotaxically切方脑块。慢慢地，完全地嵌入在安装介质的大脑。放置到切片机头部和部分采样频率使用预先确定的参数部分的大脑夹头。 </li><li>一旦组织块已经详尽的切片和部分成​​井，板盖盖子，用封口膜包装的盖子，并在-20 ° C冰箱的地方。日志总数为每个系列，部分采样间隔为每个动物的数量，以及一系列的部分。此日志将是至关重要的的轨道，保持大脑银行随后的部分去除，为今后的免疫组织化学。 </li></ol>第4部分：代表性的成果： 在这种方式的系统化采样已在我们的实验室在过去3年一个标准的做法。我们有一个很大的成功执行免疫组织化学材料已在抗原存储保存三年后，这是没有信号的恶化（图1）切片。此外，由于我们的大脑银行的一部分，我们已经接近20000系统的部分非人类记录灵长类动物的大脑（图2）作为我们的长期研究计划的一部分。 <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> 图1，这是一段从非人类灵长类动物枕极免疫染色显示NeuN第六层和间质白质神经元。这种特殊的部分是存储中的抗原，在-20 ° C保存期为2年。比例尺=100μm的。 <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="图2" /> 图2，现 ​​在我们vervet大脑银行的近20,000名来自超过30只猴子的系统部分组成。

<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.

系统抽样是一种廉价的方法，旨在最大限度地提高研究材料。此取样策略的目的是，遵守的规则，需要整个地区的系统采样的无偏体视。至关重要的是，每个系列的部分路段秩序维持。在我们的实验室中，我们已经成功地使用银行仓鼠和非人类灵长类动物的脑切片这种方法。到目前为止，我们已经收集了接近20000 vervet猴（Chlorocebus aethiops sabeus）的脑切片，进行常规免疫组已存放了一年多的?...

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brain banking: making the most of your research specimens

无偏体视学是一种准确，有效地估算总神经元的数量（或其他类型的细胞）在特定地区的利益的方法<sup&gt; 1</sup&gt;。要实现这一目标6-10探测系统部分应涵盖整个结构。通常情况下，这涉及到处理的1 / 5的部分叶片大量未处理的材料。为了最大限度地利用材料，我们提出了一个便宜的方法保留作为一个长期的存储研究计划的一部分的固定的组织。作为组织切片和处理所需的染色或抗体，替代部分应系统地放置在抗原在-20 ° C保存以备将来处理。利用24孔板，部分可以放置，以便将来检索。使用这种方法，组织可以存储和近两年的过程中的免疫组织化学处理。

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

脑银行和生物材料的系统抽样提供无偏体视学的基础上，并最大限度地从每个试样获得潜在的数据。

脑银行业：使你的研究样本中，大部分

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 次观看.  University of Montreal. 脑银行和生物材料的系统抽样提供无偏体视学的基础上，并最大限度地从每个试样获得潜在的数据。

视频: 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.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

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

科研

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.

在体内的单细胞电穿孔内完整的大脑发育

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.

膜片吸液管和夏普电极与一个可编程的拖轮

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.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

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.

使磁共振成像儿戏 - 小儿神经影像学的协议，指引及程序

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.

初压雌核发育二倍体的斑马鱼

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.

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

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.

获取标本减慢，加速和反老化的蜜蜂型号

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.

从大鼠的侧尾静脉血液取样

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: 三维脂肪组织成像的组织清除方法

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