我们非常感谢志愿扬声器的声音安德斯Bovin提供我们的电影音乐和迈克尔埃利奥特股市。提供了支持卡尔Trygger基金会战略研究计划农业功能基因组学在瑞典农业科学院，瑞典研究理事会（VR）的大学（AgriFunGen），瑞典研究理事会和环境，农业科学，空间规划（FORMAS ）。

<ol>
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两方面的P.冷杉协议进行了优化比较的A.拟南芥/ B油菜协议，治疗核糖核酸酶和蛋白酶K的浓度。的RNase去除背景信号，从而增加了信号的特异性17。蛋白酶K的治疗是需要通透的组织，使探针可以进入和杂交利益的RNA池。 DAL13反义探针了一个更高的信号无RNase的治疗（图3C，E，G）相比，用RNase处理的部分为30分钟（图2D，F和H） 。由于核糖核酸治疗没有增强信号，这是从协议中删除。蛋白酶K的治疗进行了测试三种不同的浓度; 1，3和5微克/毫升。在P 的情况下冷杉一个低或无信号，观察使用1μg/ ml的蛋白酶K（图3C - D）。一个强烈的信号，两个3微克/毫升（图3E - F）和5微克/毫升（图3G - H）的蛋白酶K由于没有明显的差异，信号强度时发现使用5微克/毫升的蛋白酶K 3微克/毫升相比，我们选择使用我们的协议在3微克/毫升。很可能需要较高的蛋白酶K含量的体育冷杉相比，雄球花的A.拟南芥和 B。 油菜花芽是由于更紧凑的组织水平较高的酚类物质和多糖。 在固定和嵌入的A.之间的一些分歧拟南芥/ B甘蓝型油菜的协议和在体育冷杉协议是显而易见的。是固定的利益的组织提供RNA的保留，这是关键的一步，因为不好固定材料可能会低或检测不到信号，即使mRNA的是非常丰富的。组织脱水，清除和嵌入式蜡在逐渐变化，以避免组织损伤，并在一些协议0.85％的NaCl添加乙醇脱水，以进一步避免收缩和肿胀的细胞 3 。嵌入过程中的脱水步骤，可在冰上进行，保持在最低RNase活性。在体育冷杉协议的4％多聚甲醛修复方案包含0.25％的戊二醛，这是应该给予更好的 RNA保留17尽管有些人认为它可能没有什么区别3。后切片材料固定到幻灯片和预处理（即脱蜡，水化，透，处理，以减少非特异性结合的探针，最后脱水）杂交前。 信号的发展，整个检测过程在这里提出的协议可在普通实验室进行，通常在1-3天，超过背景信号之间的比例可以不断地进行监测。辛标记的探针通常会更鲜明的信号相比，放射性标记的探针，更稳定，可在连续的实验重用。另一方面，灵敏度降低相比，17个放射性探针原位杂交检测表达一定的mRNA的具体使用探针。射频标签是一个强大的和敏感的标记方法，但它需要处理的放射性物质，它需要几个星期前可检测的结果。另一方面抗原标记的探针，更稳定，危险性较小，需要接触到一个只有17几天的检测介质。因此，抗原标记方法是目前的主导。最关键的一步，不管协议是探头的设计。应谨慎，以确保探针具有高特异性和，是与高品质UTPs标记的。有时，杂交温度和/或反应的长度必须为特定的探头调整。 修改后的协议，基于高辛标记探针将促进mRNA的表达本地化， 同时在从A不等的物种拟南芥到 P 冷杉 ，应稍作调整在其他植物中使用。该协议，雄球花旁，也进行了测试在不同阶段的营养芽和合子和体细胞胚胎从P.冷杉与良好的结果（未公布结果;高本汉等 ）。

<table><tbody><tr><td>Acetic anhydride</td><td>Sigma-Aldrich</td><td>A6404-200ml</td><td>minimum 98%</td></tr><tr><td>Acrylamide, linear</td><td>Ambion</td><td>9520</td><td></td></tr><tr><td>Anti-DIG-antibodies</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Blocking reagent</td><td>Roche Group</td><td>11 175 041 910 or 11 096 176 001</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030-50g</td><td>minimum 98%</td></tr><tr><td>Deionized formamide</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Denhardts solution</td><td>Sigma-Aldrich</td><td>D2532-5ml</td><td></td></tr><tr><td>DEPC, diethylpyrocarbonate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Dextran sulfate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>DIG RNA Labeling Kit (SP6/T7)</td><td>Roche Group</td><td>11175025910</td><td></td></tr><tr><td>Glutaraldehyde (25%)</td><td>Histolab</td><td></td><td></td></tr><tr><td>Glycogen</td><td>Ambion</td><td>9510G</td><td></td></tr><tr><td>Histoclear II</td><td>Histolab</td><td></td><td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td></tr><tr><td>Histowax</td><td>Histolab</td><td></td><td>Histowax or Paraplast can be used for embedding.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-500g</td><td></td></tr><tr><td>Paraplast plus</td><td>Sigma-Aldrich</td><td>P3683-1kg</td><td>Histowax or Paraplast can be used for embedding.</td></tr><tr><td>Phusion&amp;#8482; High-Fidility DNA Polymerase</td><td>Finnzymes</td><td></td><td></td></tr><tr><td>Probe-on Plus slides</td><td>Fisher Scientific</td><td>22-230-900</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308-5mg</td><td></td></tr><tr><td>RNase A</td><td>Sigma-Aldrich</td><td>R5503-100mg</td><td></td></tr><tr><td>Tissue-clear</td><td>Sakura Finetek</td><td></td><td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td></tr><tr><td>Triethanolamine</td><td>Sigma-Aldrich</td><td>T1377-100ml</td><td>minimum 98%</td></tr><tr><td>Triton X-100</td><td>use your standard product in the lab</td><td></td><td>Use one or both of Triton X-100 and Tween-20.</td></tr><tr><td>tRNA</td><td>Sigma-Aldrich</td><td>83853-100mg</td><td></td></tr><tr><td>Tween-20</td><td>use your standard product in the lab</td><td></td><td>Use one or both of Triton X-100 and Tween-20.</td></tr><tr><td>Western Blue</td><td>Promega Corp.</td><td></td><td></td></tr></tbody></table>

non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species

高通量表达分析技术提供给科学家溢出的表达谱，但他们在组织特异性表达的决议，是因为在剖析个别组织的问题有限。表达数据需要进一步证实，表达用原位杂交，采用的技术，例如本地化细胞特异性mRNA的表达模式与补充。 原位杂交方法是费力，费时，往往需要广泛的优化取决于物种和组织。 原位实验木本物种，如针叶树挪威云杉（云杉冷杉）执行比较困难。在这里， 我们目前在修改后的辛原位杂交协议，这是快速，适用的植物种类广泛 ，包括体育冷杉 。短短几年的调整，包括改变的核糖核酸酶治疗和蛋白酶K的浓度，我们可以使用该协议的研究组织特异性表达的同源基因在男性生殖器官之一裸子植物和两个被子植物 ; P。 冷杉属，拟南芥和甘蓝型油菜 。该协议工作同样适用物种和基因研究。AtAP3和BnAP3螺纹A 中的第二个和第三个花器官观察拟南芥和 B。 油菜和DAL13 P.雄球花microsporophylls 冷杉 。 为 P 冷杉蛋白酶K的浓度，用于permeablize组织，必须增加至3克/毫升，而不是1克/毫升，可能是由于更紧凑的组织和更高水平的酚类物质和多糖。所有种类的核糖核酸酶治疗被删除，由于没有相应增加特异性信号强度降低。通过比较同源基因的组织特异性表达模式，从开花植物和针叶树，我们证明了DIG原位协议提出，只有分钟的调整，可应用于广泛的植物物种。因此，这个协议避免了双方广泛的物种特定的优化和赞成DIG标记的探针的放射性标记的探针费力的使用。我们已经选择了，说明在我们的电影协议的技术要求高的步骤。 高本汉安娜和珍妮卡尔松同等贡献这项研究。 通讯作者：安娜Anna.Karlgren @ ebc.uu.se和Jens楼松德斯特伦Jens.Sundstrom @ vbsg.slu.se高本汉

Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

我们描述了一个修改的辛<em&gt;原位</em&gt;杂交协议，它是快速和适用的植物种类广泛，包括挪威云杉。短短几年的调整，包括改变的核糖核酸酶治疗和蛋白酶K的浓度，该协议可用于在不同的组织和物种的研究。

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

non-radioactive-situ-hybridization-protocol-applicable-for-norway

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

20.9K 次观看.  Uppsala University. 我们描述了一个修改的辛原位杂交协议，它是快速和适用的植物种类广泛，包括挪威云杉。短短几年的调整，包括改变的核糖核酸酶治疗和蛋白酶K的浓度，该协议可用于在不同的组织和物种的研究。

视频: Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

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 ...

科研

神经科学

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

环境科学

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

免疫与感染

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

发育生物学

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

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

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

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 ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

生物学

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization (WMISH) is a common technique used for visualizing the location of expressed RNAs in embryos. In this process, synthetically produced RNA probes are first complementarily bound, or "hybridized," to the transcripts of target genes. Immunohistochemistry or fluorescence is then used to detect these RNA hybrids, revealing spatial and temporal patterns of gene expression. Unlike traditional in situ hybridization techniques, which require thin tissue sections whose images will need to be computationally reassembled, the whole-mount technique allows gene expression patterns to be assessed over the entire embryo or structure.
This video will introduce the basic concepts of whole mount staining and detail key procedural steps, including probe design and production, embryo fixation and staining, and post-hybridization signal detection. Viewers will then learn about how developmental biologists are applying WMISH to current research studies.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

摘要

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此视频中的章节

双荧光原位杂交食脑切片

就地石蜡镶嵌成人珊瑚样品的杂交技术

荧光

阿斯蒂亚纳克斯胚胎的全原位杂交

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色

结合双荧光

原位杂交技术在植物中的精确定位成绩单

放射性的原位杂交检测组织不同的基因表达模式

混合式切割:用于顽拗植物组织样品的改进切法

整个装载原位杂交

非放射性原位挪威云杉杂交议定书适用的植物种类范围

摘要

探索更多视频

此视频中的章节

双荧光原位杂交食脑切片

就地石蜡镶嵌成人珊瑚样品的杂交技术

荧光

阿斯蒂亚纳克斯胚胎的全原位杂交

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

结合双荧光

原位杂交技术在植物中的精确定位成绩单

放射性的原位杂交检测组织不同的基因表达模式

混合式切割:用于顽拗植物组织样品的改进切法

整个装载原位杂交

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色