Name: a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis
Uploaded: 2016-03-15T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

وأيد هذا العمل من خلال تمويل لDJJ من خلال مشروع DFG # JA2108 / 2-1.

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الطريقة الموصوفة هنا يسمح التصور الفعال للنسخ الرنا مع فرضا متفاوتة مستويات التعبير في جميع مراحل تطور المنقعية الراكدة. لإزالة الأجنة واليرقات من كبسولات بهم ونحن محاكمتهم مجموعة متنوعة من المواد الكيميائية، صدمة التناضحي والمعالجات الفيزيائية وذكرت لencapsulat...

الرخويات هي مجموعة من الحيوانات التي تحمل الفائدة من تنوع واسع من التخصصات العلمية. وعلى الرغم من تنوع الصرفي من 1، ثراء الأنواع (في المرتبة الثانية بعد المفصليات من حيث عدد الأنواع 2) وصلة إلى مجموعة واسعة من التجارية 3، 4 الطبية والعلمية القضايا 5-8، هناك عدد قليل نسبيا من الأنواع الرخويات التي يمكن أن تدعي ل يكون كلا النموذجين علمية مجهزة تجهيزا جيدا وسهلة للحفاظ في بيئة معملية. واحد الرخويات التي تستخدم كثيرا من قبل التخصصات مثل علم الأعصاب 9، والسمية الإيكولوجية 10 وأكثر في الآونة الأخيرة البيولوجيا التطورية 11،12، هو المنقعية الراكدة، وذلك أساسا بسبب التوزيع على نطاق واسع وسهولة القصوى من الصيانة. وعلى الرغم من شعبيته بوصفه الكائن &#39;نموذج&#39; وتاريخها الطويل من الاستخدام من قبل علماء البيولوجيا التطورية 13-19، ومجموعة وقوة الأدوات الجزيئية المتاحة للL. stagnيكمن المجتمع العلمي أليس بعيدا وراء ذلك من مزيد من النماذج التقليدية الحيوان (ذبابة الفاكهة، والماوس، قنفذ البحر والديدان الخيطية). لدينا الرغبة في تطوير المنقعية كما ينبع نموذج جزيئي من مصلحة في الآليات الجزيئية التي توجه تشكيل قذيفة. هذا دفعنا لصقل مجموعة من التقنيات التي من شأنها أن تسمح لتصور فعال، بما يتفق وحساسة في التعبير الجيني خلال تطوير المنقعية الصورة. جبل بأكمله في الموقع التهجين (WMISH) يعمل على نطاق واسع لمجموعة متنوعة من الكائنات الحية النموذج والتي تم فيها استخدام لأكثر من 40 عاما 20. في وجوهها المختلفة، ISH يمكن استخدامها في توطين مكانيا مواضع محددة على الكروموسومات، الريباسي، مرنا والرنا الصغير. واحدة من التحديات التي تحتاج إلى معالجة قبل صقل طريقة WMISH لL. كان الراكدة قضية استخراج بلطف وكفاءة الأجنة واليرقات من مراحل متفاوتة من رانه كبسولات البيض التي يتم إيداعها. هذا الاستخلاص، أو &quot;نزع المحفظة&quot;، يحتاج إلى أن يتحقق بكفاءة من أجل جمع المادية الكافية لالواردة في تجربة الموقع، وفي الوقت نفسه الحفاظ على سلامة المورفولوجية والخلوية. بينما الكائنات النموذج الأخرى أيضا الخضوع لتطوير مغلفة، في أيدينا يمكن أن تستخدم أيا من الأساليب ذكرت لتلك الأنواع بنجاح في L. الراكدة. وبالتالي فإن الأهداف العامة لهذه الطريقة هي: لاستخراج L. الراكدة الأجنة واليرقات من كبسولات بها بطريقة الإنتاجية العالية، لتطبيق علاجات ما قبل التهجين أن تحسين إشارة WMISH، لإعداد الأجنة واليرقات مع WMISHsignals مرضية للتصوير.

<table><tbody><tr><td>Featherweight forceps</td><td>Ehlert &amp;amp; Partner</td><td>#4181119</td><td></td></tr><tr><td>Silicon tubing</td><td>Glasger&amp;auml;tebau OCHS GmbH</td><td>760070</td><td></td></tr><tr><td>Glass capillaries</td><td>Hilgenberg</td><td>1403547</td><td></td></tr><tr><td>12 well tissue culture dishes</td><td>Carl Roth</td><td>CE55.1</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Carl Roth</td><td>P733.1</td><td>CAUTION - May cause cancer. Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>Carl Roth</td><td>CN06.3</td><td>CAUTION - CAUSES EYE IRRITATION. MAY CAUSE RESPIRATORY TRACT AND SKIN IRRITATION. Avoid breathing dust. Avoid contact with eyes, skin and clothing. Use only with adequate ventilation</td></tr><tr><td>Magnesium Chloride</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>Tween-20</td><td>Carl Roth</td><td>9127.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium Chloride</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>Ficoll type 400</td><td>Carl Roth</td><td>CN90.1</td><td></td></tr><tr><td>polyvinylpyrrolidone K30 (MW 40)</td><td>Carl Roth</td><td>4607.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Nuclease freeBovine Serum Albumin</td><td>Carl Roth</td><td>8895.1</td><td></td></tr><tr><td>Salmon sperm</td><td>Carl Roth</td><td>5434.2</td><td></td></tr><tr><td>Heparin</td><td>Carl Roth</td><td>7692.1</td><td>CAUTION - ADVERSE EFFECTS INCLUDE HEMORRHAGE, LOCAL IRRITATION. POSSIBLE ALLERGIC REACTION IF INHALED, INGESTED/CONTACTED. EYES/SKIN/RESPIRATORY TRACT IRRITANT. POSSIBLE HYPERSENSITIZATION. DURING PREGNANCY HAS BEEN REPORTED TO INCREASE RISK OF STILLBIRTH</td></tr><tr><td>Proteinase-K</td><td>Carl Roth</td><td>7528.1</td><td></td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3790.2</td><td></td></tr><tr><td>Deionised formamide</td><td>Carl Roth</td><td>P040.1</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Standard formamide</td><td>Carl Roth</td><td>6749.3</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Triethanolamine</td><td>Carl Roth</td><td>6300.1</td><td>CAUTION - Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling.</td></tr><tr><td>Acetic anhydride</td><td>Carl Roth</td><td>4483.1</td><td>CAUTION - CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT</td></tr><tr><td>Maleic acid</td><td>Carl Roth</td><td>K304.2</td><td>CAUTION - Very hazardous in case of eye contact (irritant), of ingestion, . Hazardous in case of skin contact (irritant), of inhalation (lung irritant). Slightly hazardous in case of skin contact (permeator). Corrosive to eyes and skin.</td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630-250ML</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. Harmful if swallowed.</td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>10,800-6</td><td>CAUTION - Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation.</td></tr><tr><td>Glycerol</td><td>Carl Roth</td><td>3783.1</td><td></td></tr><tr><td>Blocking powder</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>Anti DIG Fab fragments AP conjugated</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Tris-HCl</td><td>Carl Roth</td><td>9090.3</td><td></td></tr><tr><td>4-Nitro blue tetrazolium chloride in dimethylformamide&amp;nbsp;</td><td>Carl Roth</td><td>4421.3</td><td>CAUTION - May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes.</td></tr><tr><td>5-bromo-4-chloro-3-indolyl-phosphate</td><td>Carl Roth</td><td>A155.3</td><td>CAUTION - Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant.&amp;nbsp;</td></tr><tr><td>N-acetyl cysteine</td><td>Carl Roth</td><td>4126.1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Carl Roth</td><td>6908.1</td><td>CAUTION - May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Tergitol</td><td>Sigma</td><td>NP40S</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium dodecyl sulphate</td><td>Carl Roth</td><td>CN30.3</td><td>CAUTION - Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation.</td></tr><tr><td>Potassium Chloride</td><td>Carl Roth</td><td>6781.1</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>4984.1</td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>3904.1</td><td></td></tr><tr><td>Tri sodium citrate dihydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;Na&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>3580.1</td><td>CAUTION - May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Mineral oil&amp;nbsp;</td><td>Carl Roth</td><td>HP50.2</td><td></td></tr><tr><td>InSituPro-Vsi&amp;nbsp;</td><td>Intavis</td><td></td><td>www.intavis.de/products/automated-ish-and-ihc</td></tr></tbody></table>

a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis

جبل بأكمله في الموقع التهجين (WMISH) هي تقنية تسمح للقرار المكاني للجزيئات الحمض النووي (في كثير من الأحيان من mRNAs) ضمن إعداد الأنسجة &quot;جبل بأكمله، أو مرحلة النمو (مثل الجنين أو يرقة) من الفائدة. WMISH هي قوية للغاية لأنها يمكن أن تساهم بشكل كبير في توصيف وظيفي من الجينوم metazoan معقدة، وهو التحدي الذي أصبح أكثر من عنق الزجاجة مع طوفان من البيانات تسلسل الجيل القادم. على الرغم من بساطة المفاهيمية للتقنية وغالبا ما يحتاج الكثير من الوقت لتحسين مختلف المعايير المتأصلة في تجارب WMISH لنظم نموذج الجديدة؛ يعني الفروق الدقيقة في خصائص الخلوية والكيمياء الحيوية بين أنواع الأنسجة والمراحل التطويرية التي أسلوب WMISH واحد قد لا يكون مناسبا لجميع الحالات. لقد قمنا بتطوير مجموعة من الأساليب WMISH لعاودت الظهور نموذج البطني المنقعية الراكدة التي تولد يتفق وإشارات WMISH واضحة لمجموعة من الجينات، وعبر جميع مراحل النمو. وتشمل هذه الأساليب احالة يرقات العمر الزمني غير معروف إلى نافذة التخلق، وإزالة فعالة للأجنة ويرقات من كبسولات البيض، وتطبيق العلاج بروتين-K المناسبة لكل نافذة التخلق، والتهجين، بعد التهجين ومناعي خطوات. وتوفر هذه الأساليب على الأساس الذي الإشارة الناتجة عن نسخة RNA معينة يمكن أن يكون مزيد من الصقل مع تعديلات محددة التحقيق (التحقيق في المقام الأول التركيز ودرجة الحرارة التهجين).

تم إنشاء أنماط تلطيخ WMISH ممثل هو مبين في الشكل (3) باستخدام تقنية الموضحة أعلاه، وتعكس مجموعة متنوعة من أنماط التعبير المكاني للجينات لها دور في مجموعة من العمليات الجزيئية بدءا من تشكيل قذيفة (الجينات رواية 1 و 2 و 3 و 4)، إلى إشارات ا...

Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

ككل جبل<em&gt; في الموقع</em&gt; طريقة التهجين لالبطني الأقدام الرخويات<em&gt; الراكدة المنقعية</em

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

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Research

JoVE Journal

Biology

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

8.8K Views.  Georg-August University of Göttingen. The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Video: A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

والطريقة موسع لل<em&gt; في الوضع الطبيعي</em&gt; تحديد النجاح في التزاوج أنثى الكركند الأمريكية (<em&gt; Homarus americanus</em&gt;)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Environment

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

التصور الحجمية في الشجاعة القراد بالجامعة-جبل في الموقع التهجين

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Genetics

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

تأمين تدفق الأحماض النووية الخلوي ومضان في الموقع التهجين (LNA تدفق FISH) : طريقة لاكتشاف الحمض النووي الريبي البكتيرية الصغيرة

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Immunology and Infection

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.

ووليماونت في الموقع التهجين للأجنة أستياناكس

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

Developmental Biology

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

جبل كله<em&gt; في الموقع</em&gt; التهجين من E8.5 لأجنة الفئران E11.5

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

إنتاجية عالية في الموقع أسلوب التهجين لتوصيف أنماط التعبير مرنا في الفأر الجنينية السفلى المسالك البولي التناسلي

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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&gt; في الموقع</em&gt; التهجين في الأجنة جبل الجامعة وعلم الأنسجة الخلية مقتبس عن Elasmobranchs البحرية

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

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

تأثير الغدة ذكر ملحق المنتجات على وضع البيض في بطني الأرجل الرخويات

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

في الموقع التهجين لسيبونكولوس nudus Coelomic السائل

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

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