作者要感谢她的专长苏康奈尔女士的协助下，在树脂嵌入和她的实验室技术女士千金墓群。作者要感谢他们的意见弗兰克Kandziora教授和博士玛丽 - 安妮Polboth。

<ol><li>该组织是放置在一个密封的容器中含有10％磷酸的福尔马林溶液3。确保尽量减少暴露在光线下，如果要执行，作为荧光光敏感荧光分析。 </li><li>建立将需要组织的方向。 </li><li>修剪大小的标本，在正确的方向，用一个带锯。确保安全措施得到遵守。试样，然后将放在不透明的容器，其中的量应该是10倍左右大小的标本，以达到充分固定。试样应保持在福尔马林溶液中的一到两个星期取决于其大小总。建议使用控制组织，用于测试和优化的目的。 </li><li>检查组织成适合嵌入模具和组织放入一个密封的不透明的容器中含有70％的乙醇。 </li><li>升序乙醇浓度的组织过程，从光线和不断搅拌下屏蔽。 <ol class="lalpha"><li> 70％的乙醇，1个星期</li><li> 80％的乙醇，1个星期</li><li> 90％的乙醇，1个星期</li><li> 100％的乙醇，1个星期</li></ol></li><li>组织正丁醇1周，然后清除，再从光，并不断搅拌下屏蔽。 </li><li> 树脂和骨骼的密度应密切配合 ，可以改变树脂的密度（见第8步），我们建议首先要保证正确的密度嵌入试样。我们用光学显微镜和荧光分析Technovit 7100（贺利氏古莎）。如果需要免疫组化是另一种树脂，必须使用如Technovit 9100（贺利氏古莎）。 </li><li> Technovit 7100溶液的制备可混合分装软化解决方案提供的套件，如果需要的话，但不超过5％的配制溶液的体积，软化解决方案在我们的经验是很少需要。无水乙醇和基液体Technovit 7100等份混合和应用的组织，作为减少光线照射24小时前渗透解决方案，并搅拌。 </li><li>渗透溶液，然后用1克固化剂，我（= 1袋），这是溶解在100毫升的基液。这取代了前渗透的解决方案是允许的渗透不断搅拌1周。 </li><li>组织，切割面正面朝下放入模具。确保有一个保证金周围组织的树脂（理想情况下至少5毫米），这确保了块强度。混合溶液的制备Technovit 7100 15mls与1毫升固化剂二，倒在模具周围的标本和至少5 millimietres覆盖。固化将在2小时内开始，但在一夜之间将完成。 </li><li>然后执行安装使用Technovit 3040（贺利氏古莎）塑造一个备份块。在两部分粉1部分液体，以获得一种粘性液体的体积比混合Technovit 3040。 Technovit 3040倒入水平至少2毫米以上的块基地块背面的凹槽。约10分钟后，模具可剥离和块准备。 </li><li>的密度，因此甲基丙烯酸树脂切割性能很容易受到室内的环境湿度。因此，应块存储在一个dessicator。 </li><li>地面切片厚度为20-50微米之间产生较大的部分。它是有用的荧光分析（见下节）较厚部分产生明亮的荧光。安全金刚石锯片macrotome。添加润滑剂，如汽油，水库上macrotome。安全钳块，定位到刀片。允许研磨发生缓慢（约20-30分钟，每节），以确保足够的润滑。第一部分公开的切割面和orientates部分，它应该被丢弃。 </li><li> Superfrost加幻灯片，然后放置在下一节。本节有一种倾向，因此，我们建议配售的超额部分三明治袋，然后拿着另一张幻灯片的地方夹住它平卷曲。 1小时摄氏60-80度之间，然后放入一个孵化器。这种柔软的丙烯酸甲酯，并帮助部分坚持在一个单位的方式幻灯片。 </li><li>可清洗地面部分和根据需要在一个温柔的方式使用细砂纸打磨。 </li><li>爬犁切片切薄片，它提供了最好的部分光microsocopy。使用雪橇切片机，还增加了刚性，这是需要切割这些硬模块。另外一个动力旋转切片机都可以使用。用锋利的不锈钢刀片。最好是有两个叶片可作为一个可用于其他削尖。刀片应该做一个45度角块。苏如果需要，应用湿纸块，可以实现rface软化。这可能需要几个部分，达到理想的切削条件下，获得优质的部分。本节，然后飘然水浴上，并在16个以上的幻灯片上。 </li><li>染色试剂列在下面的材料。协议是基于班克罗夫特4等。由于厚度的变化，建议以优化测试幻灯片的污渍。机架染色可能会导致组织浮动，所以，可能需要添加的污渍直接向扁平幻灯片的幻灯片。相同的协议或机架染色染色组织形态计量学要求的结果一致是必要的。 <ol class="lalpha"><li>苏木和曙红<ol class="lroman"><li>染色Haematoxyilin 5-10分钟</li><li>斯科特小号自来水冲洗</li><li>染色与曙红5分钟</li><li>自来水洗</li><li>在二甲苯清除</li><li>挂载</li><li> 结果 <ol><li>骨样=粉红色</li><li>钙化骨=紫褐色</li><li>核=蓝色</li></ol></li></ol></li><li>冯科萨<ol class="lroman"><li>放入硝酸银溶液和矿化骨，直到变成黑色，暴露在强烈的光线（约10分钟） </li><li>在蒸馏水洗三次</li><li> 5分钟与硫代硫酸钠的威胁</li><li>蒸馏水洗净</li><li>染液与Safrinin Ø </li><li>在二甲苯清除</li><li>挂载</li><li> 结果 <ol><li> Mineralised骨=黑色</li><li>骨样=红/粉红色</li></ol></li></ol></li><li>马森Goldner小号三色<ol class="lroman"><li>碱性酒精清洗（90mls 80％的乙醇和25％的氨水20分钟10mls） </li><li>用水冲洗</li><li>蒸馏水冲洗</li><li>与Weigert小号苏木染色10分钟</li><li>蒸馏水冲洗</li><li>染色与丽春红，品红最终解决5分钟</li><li>用1％醋酸冲洗15秒</li><li>磷钼酸橙色G解决方案5分钟染色</li><li>用1％醋酸冲洗15秒</li><li>染色浅绿色5分钟</li><li>用1％醋酸冲洗3变化</li><li>蒸馏水冲洗</li><li>挂载</li><li> 结果 <ol><li> Mineralised骨=绿色</li><li>骨样=红/橙</li><li>核=蓝灰色</li><li>软骨=紫色</li></ol></li></ol></li></ol></li><li>荧光染料的准备工作： </li><li>这些管理缓慢静脉注射，相隔至少两个星期。 </li><li>剂量<ol class="lalpha"><li>钙黄绿素绿10毫克/公斤IV </li><li>土霉素50毫克/公斤IV </li><li>茜素络合剂30毫克/公斤IV </li></ol></li><li>钙黄绿素绿色的制备<ol class="lalpha"><li> 1克钙黄绿素绿色的是用1 M NaOH溶液滴定，直至被解散为止。 </li><li>用1％NaOH调整pH值至pH = 7.1-7.2。 </li><li>光屏蔽过​​滤器，并保持</li></ol></li><li>茜素络合剂的制备<ol class="lalpha"><li>茜素络合克用1 M NaOH溶液滴定，直至解散。 </li><li> pH值，然后用1％盐酸或氢氧化钠调整至pH = 7.1-7.2。 </li><li>光屏蔽过​​滤器，并保持</li></ol></li><li>土霉素的制备<ol class="lalpha"><li>土霉素是作为一个现成的抗微生物药物。无需配制。 </li></ol></li></ol>

<ol>
	<li>Kerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Functional Histology. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 2, 595-610 (1987).</li><li>An, Y. H., Martin, K. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of histology methods for bone and cartilage. , (2003).</li><li>Bancroft, J. D., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and Practice of Histological Techniques. , 352-360 (2008).</li></ol>

溶剂： <ul><li>酸，10％的正常缓冲福尔马林，乙醇，二甲苯和丁醇 所有组织学级，无硫。 Grale科学Pty有限公司 http://www.grale.com.au </li></ul> 模具： <ul><li>剥离，塑料模具 Grale科学</li></ul> 幻灯片： <ul><li> SUPERFROST PLUS的显微镜幻灯片 博士伦科学Pty有限公司 <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> 树脂： <ul><li> GlycolMethacrylate Technovit 7100 贺利氏古莎 <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a> </li><li>背座树脂Technovit 3040 HISTO块 贺利氏古莎</li></ul> 渍 （Grale科学试剂） Goldner三色染色</li> 1。 Weigert氏铁hemotoxylin（DeGroat） <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A）Hemotoxylin解决方案</td></tr><tr><td> Hemotoxylin </td><td> 1克</td></tr><tr><td>无水酒精</td><td> 100毫升</td></tr><tr><td colspan="2" style="font-weight:bold;"> B）铁溶液</td></tr><tr><td> 30％三氯化铁水溶液</td><td> 4ML </td></tr><tr><td>盐酸（浓） </td><td> 1ML </td></tr><tr><td>蒸馏水</td><td> 95ml置于</td></tr></table>过滤和添加到同等体积的hemotoxylin立即前染色使用。 2。丽春红，品红azophloxin股票解决方案<table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A）丽春红 - 二甲基苯胺的解决方案</td></tr><tr><td>丽春红 - 二甲基苯胺</td><td> 0.75克</td></tr><tr><td>酸fuchsion </td><td> 0.25克</td></tr><tr><td>醋酸</td><td> 1ML </td></tr><tr><td colspan="2">混合，并添加蒸馏水（100毫升） </td></tr><tr><td colspan="2" style="font-weight:bold;"> B）azophloxin解决方案</td></tr><tr><td> Azophloxin </td><td> 0.5克</td></tr><tr><td>乙酸</td><td> 0.6mL </td></tr><tr><td colspan="2">混合，并添加蒸馏水（100毫升） </td></tr><tr><td colspan="2" style="font-weight:bold;"> c）最后的工作染色解决方案</td></tr><tr><td>丽春红，品红溶液</td><td> 5 - 10毫升</td></tr><tr><td> Azophloxin </td><td> 2ML </td></tr><tr><td> 0.2％的醋酸溶液</td><td> 88mL </td></tr></table> 3。浅绿色解决方案<table style="margin-left:40px;" border="0"><tr><td>浅绿色</td><td> 1克</td></tr><tr><td>乙酸</td><td> 1ML </td></tr><tr><td colspan="2">混合，并添加蒸馏水（500毫升） </td></tr></table> 4。 Phophomolybdic酸性橙G解决方案<table style="margin-left:40px;" border="0"><tr><td>磷钼酸</td><td> 3克</td></tr><tr><td>橙G </td><td> 2克</td></tr><tr><td colspan="2">溶解在500ml的蒸馏水，加​​冰糖麝香草酚。 </td></tr></table>冯科萨染色<table style="margin-left:40px;" border="0"><tr><td> 1％硝酸银水溶液</td></tr><tr><td> 2.5％硫代硫酸钠</td></tr><tr><td> 1％番红O </td></tr></table>阿尔辛蓝染色<table style="margin-left:40px;" border="0"><tr><td>阿尔新蓝8GX </td><td> 1克</td></tr><tr><td> 3％醋酸溶液</td><td> 100毫升</td></tr></table> Hemotoxylin和曙红染色<table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A）哈里斯的hemotoxylin </td></tr><tr><td> Hemotoxylin </td><td> 2.5克</td></tr><tr><td>无水酒精</td><td> 25ML </td></tr><tr><td>明矾</td><td> 50克</td></tr><tr><td>蒸馏水</td><td> 500毫升</td></tr><tr><td>氧化汞</td><td> 1.25克或</td></tr><tr><td>碘酸钠</td><td> 0.5克</td></tr><tr><td>冰醋酸</td><td> 20ML </td></tr><tr><td colspan="2" style="font-weight:bold;"> B）曙红溶液</td></tr><tr><td colspan="2">使用商业化生产的曙红。 </td></tr></table>所有染色的染料粉末Gurr染料粉末。 所有试剂均为分析纯，而不是实验室试剂 荧光 <table border="1" cellspacing="0" cellpadding="0"><tr><td> 制造商</td><td>荧光</td><td>目录编号</td></tr><tr><td> Sigma - Aldrich公司，悉尼</td><td>钙黄绿素绿</td><td> C0875 </td></tr><tr><td> Sigma - Aldrich公司，悉尼</td><td>茜素络合剂</td><td> A3882 </td></tr><tr><td>英特威澳大利亚，维多利亚</td><td>土霉素 （现成的抗菌药物） </td><td>商标名称; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

脱钙骨组织学显示骨微架构。它同时显示矿化骨，它提供了对骨代谢或骨形成和骨吸收的重要信息和细胞成分。这具有巨大的重要性在各种不同的临床和研究中的应用。它会产生美丽的图像技术，如荧光评估和组织形态计量学2 1和允许。荧光分析是一种钙结合的荧光染料注射在特定的时间点，在那个特定的时间允许的矿化量的量化技术。组织形态计量学是在微观层面的骨定量过程。 执行脱钙骨组织学是在技术上具有挑战性，特别是大尺寸标本。它要求在技术标准石蜡嵌入组织学的变化。该视频演示了质量好的部分生产过程和演示的技术难点和方法，克服这些。实现方式类似于其他软组织的密度和骨的通透性较低，需要相当长的固定和处理时间，但是由于经常服用几个星期，试样制备，固定和处理。嵌入使用类似或等于硬度和密度，如甲基丙烯酸甲酯树脂骨支撑介质，但不像石蜡渗透和嵌入，这是一个不可逆转的步骤。切片可以通过研磨产生较厚的部分，这是最适用于如荧光分析的研究。这是最好的实现上macrotome使用的钻石刀片。另外，较薄，可产生光镜，这是一个非常锋利的刀片使用大锤切片。爬犁切片提供了额外的的大，硬模块所需的强度和稳定性。树脂包埋切片可染各种各样的污渍，这是证明。

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

脱钙骨组织学各种临床和研究中的应用提供了重要信息。这在技术上是具有挑战性，特别是大尺寸的标本。该视频演示了质量好的部分生产过程和演示的技术难点和方法，克服这些。

脱钙骨组织学，形态计量学和荧光染料分析制备

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Nanyang Technological University

Research

JoVE Journal

Biology

27.1K 次观看.  Monash University. 脱钙骨组织学各种临床和研究中的应用提供了重要信息。这在技术上是具有挑战性，特别是大尺寸的标本。该视频演示了质量好的部分生产过程和演示的技术难点和方法，克服这些。

视频: Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

骨条件培养基的制备和生物测定

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...