Name: double fluorescence in situ hybridization in fresh brain sections
Uploaded: 2010-08-14T17:00:00
Description: Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

NIH / NIDCD和施密特基金会到RP的赠款支持的工作。

该协议是基于标准的放射性和非放射性原位杂交方法，以前我们和其他人开发的检测在脑组织1-7中的一个或两个成绩单物种的发展和完善。下面所描述的协议有2或3天的总长度，这取决于由最终用户选择的程序中断的数量。下面详细的所有步骤都riboprobe杂交步骤和杂交后洗外，在室温下进行。在此方法中涉及的所有步骤所需的解决方案和缓冲区，可以发现在此协议结束。 <p class="...

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我们使用这个协议来研究脊椎动物的大脑是如何neurochemically和功能组织，并确定如何行为相关的感官刺激的影响在成人大脑 8-10元基因组机械。我们已经成功地使用这种方法在小鼠，大鼠和鸣禽脑组织，但预计此协议将很容易适应从脊椎动物物种的阵列，或许非神经组织得到的脑切片。需要获得可靠的结果和最佳的信号的关键控制已经详细广泛，本集团其他地方11，12 。

解决方案<ol><li>列洗涤缓冲液：10毫米的Tris - HCl，0.15 M氯化钠（NaCl），0.05毫米EDTA，50微克/μL的tRNA，0.1％SDS于50 ml DEPC处理过的水。 </li><li>列封闭液：10毫米的Tris - HCl 0.1毫米，50毫米氯化钠，EDTA于50 ml DEPC处理过的水。 </li><li> TNT缓冲液：60毫升的Tris - HCl，1米18毫升5 M氯化钠和1.8毫升的Triton X - 100的DEPC处理过的水在600毫升。 </li><li> TNB的缓冲区：100毫米的Tris - HCl，8.3微克/μLBSA，0.15 M氯化钠和3％的Triton X - 100的DEPC处理过的水。 </li><li> TE缓冲液：10毫米的Tris - HCl，pH值7.5加1 mM的EDTA，pH值7.5。 </li><li>乙酰化的解决方案：三乙醇胺加DEPC处理过的水200毫升醋酸酐0.5毫升2.7毫升。 </li><li>杂交液：50％甲酰胺，SSPE的2倍，2微克/μL的tRNA，1μg/μL的BSA和1微克/微升聚在DEPC处理的水一个。它需要每节16升。 </li></ol>

double fluorescence in situ hybridization in fresh brain sections

在这里，我们描述了一个双荧光修改后的版本<em&gt;原位</em优化杂交（dFISH）方法检测mRNA的新鲜冰冻脑切片的兴趣。我们的小组已经成功地使用这种方法，研究基因的共同监管。更具体地说，我们使用的dFISH方法，探讨组织的解剖，神经化学物质的属性，和感官体验在中央感官电路的影响，在单细胞分辨率。在小鼠，大鼠和鸣禽脑组织，但该协议已经得到验证，预计将很容易适应其他脊椎动物物种，以及非神经组织的数组。在这部影片中，我们提供详细演示了此过程的主要步骤。

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

该协议涉及非放射性<em&gt;原位</em&gt;杂交程序，使在脊椎动物的大脑薄片，同时识别两个种成绩单，在一个单细胞的决议。

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

科研

神经科学

Morphometric Analyses of Retinal Sections

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...