אנו מודים מאוד אנדרס Bovin שסיפק את המוזיקה לסרט שלנו על מניות מייקל אליוט להתנדבות להיות הקול הדובר. תמיכה סופק על ידי קרל Trygger קרן, תוכנית אסטרטגית המחקר Genomics פונקציונלית חקלאי (AgriFunGen) באוניברסיטת שוודית למדעי החקלאות, מועצת המחקר השוודי (VR), ומועצת המחקר השוודי לאיכות הסביבה, למדעי החקלאות, תכנון מרחבי (FORMAS ).

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeotic+gene+APETALA3+of+Arabidopsis+thaliana+encodes+a+MADS+box+and+is+expressed+in+petals+and+stamens.">The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.</a> Cell. 68 (4), 683-697 (1992).</li><li>Bowman, J. L., Smyth, D. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directing+flower+development+in+Arabidopsis.">Genes directing flower development in Arabidopsis.</a> Plant Cell. 1 (1), 37-52 (1989).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399 (6732), 144-148 (1999).</li><li>Sundstrom, J., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conifer+reproductive+development+involves+B-type+MADS-box+genes+with+distinct+and+different+activities+in+male+organ+primordia.">Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.</a> Plant J. 31 (2), 161-169 (2002).</li><li>Carlsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+altered+expression+of+a+large+number+of+nuclear+genes+in+developing+cytoplasmic+male+sterile+Brassica+napus+flowers.">Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers.</a> Plant J. 49 (3), 452-462 (2007).</li><li>Teixeira, R. 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שני היבטים של פ אשוח פרוטוקול היו אופטימיזציה לעומת A. thaliana / B. napus פרוטוקול, RNase טיפול וריכוז proteinase K. RNase מסיר אותות רקע ובכך מגביר את האות 17 סגוליות. הטיפול K proteinase יש צורך permeabilize הרקמות כך לחקור את יכולה להיכנס לבריכה להכליא RNA של עניין. Antisense DAL13 בדיקה נתן את האות גבוה ללא RNase הטיפול (איור 3C, E, G) לעומת סעיפים שטופלו RNase למשך 30 דקות (איור 2 ד, F ו-H). מאז RNase טיפול לא לשפר את האות הוא הוסר מן הפרוטוקול. הטיפול K proteinase נבדקו בשלושה ריכוזים שונים: 1, 3 ו - 5 מיקרוגרם / מ&quot;ל. במקרה של פ אשוח אות נמוך או לא נצפתה באמצעות 1 מיקרוגרם / מ&quot;ל proteinase K (איור 3C-D). איתות חזק הושג עם 3 מיקרוגרם הן / מ&quot;ל ​​(איור 3E-F) 5 ​​מיקרוגרם / מ&quot;ל ​​(איור 3G-H) proteinase ק מאז שום הבדל ניכר עוצמת האות נמצא בשימוש 5 מיקרוגרם / מ&quot;ל ​​proteinase K לעומת 3 מיקרוגרם / מ&quot;ל, בחרנו להשתמש 3 מיקרוגרם / מ&quot;ל ​​בפרוטוקול שלנו. סביר להניח כי הצורך של ריכוז גבוה proteinase K ב פ האצטרובלים הזכריים אשוח לעומת A. thaliana ו-B ניצנים napus פרחוני בשל רקמת יותר קומפקטי עם רמות גבוהות יותר של phenolics ופוליסכרידים. במהלך קיבעון והטבעה כמה הבדלים בין א &#39; thaliana / B. napus פרוטוקול פ אשוח פרוטוקול ניכרים. הרקמה עניין קבוע לספק שמירה רנ&quot;א זה הוא שלב קריטי שכן חומרים קבועים גרוע עשוי לתת אות נמוכה או בלתי מורגשת אף mRNA הוא נרחב ביותר. רקמה היא התייבשות, מפונה מוטבע שעווה שינויים הדרגתיים כדי למנוע נזק לרקמות, ובחלק NaCl פרוטוקולים 0.85% מתווסף אתנול של התייבשות נוספת למנוע הצטמקות ונפיחות של התאים 3. צעד התייבשות במהלך הטבעה יכול להתבצע על קרח כדי לשמור על פעילות RNase לפחות. ב פ אשוח פרוטוקול לתקן את 4% פתרון paraformaldehyde מכיל glutaraldehyde 0.25%, שאמור לתת שימור טוב יותר RNA 17 אף כי יש הטוענים כי זה כנראה לא ישנה 3. לאחר חתך את החומר הוא קבוע על גבי שקופיות pretreated (כלומר dewaxed, rehydrated, permeabilized, טיפול להפחתת הלא ספציפית מחייב בדיקה ומיובש סוף סוף) לפני הכלאה. בפרוטוקול המובא כאן הליך זיהוי כל יכול להתבצע במעבדות רגילות, אות מתפתחת בדרך כלל בתוך 1-3 ימים ואת היחס בין אות על רקע יכול להיות במעקב רציף. DIG שכותרתו בדיקות בדרך כלל לתת איתות מובהק יותר לעומת בדיקות שכותרתו רדיואקטיבי, יציבים יותר, וניתן לעשות בה שימוש חוזר בניסויים עוקבים. מצד שני, רגישות מופחתת לעומת בדיקות רדיואקטיבית 17. הכלאה In situ מזהה ביטוי באמצעות תוויות בדיקה ספציפית mRNA מסוים. רדיו תיוג היא שיטת תיוג חזקים רגיש אבל זה דורש טיפול של רדיואקטיביות וזה לוקח כמה שבועות לפני התוצאות ניתן לאתרם. Antigen שכותרתו בדיקות מצד שני, הם יציבים יותר, מסוכנים פחות דורשים חשיפה בינונית גילוי לכמה ימים רק 17. לפיכך, אנטיגן תיוג שיטות כרגע שולט. השלב הקריטי ביותר ללא תלות בפרוטוקול הוא העיצוב בדיקה. יש להקפיד על מנת לוודא בדיקה יש סגוליות גבוהה התווית UTPs באיכות גבוהה. לפעמים הטמפרטורה הכלאה ו / או אורך התגובה צריך להיות מותאם בדיקות ספציפיות. פרוטוקול שונה שלנו מבוסס על DIG שכותרתו בדיקות יקל לוקליזציה של ביטוי mRNA בו זמנית מינים החל א thaliana ל פ אשוח, ויש עם שינויים קלים להיות שמיש בכל מיני צמחים אחרים. יש פרוטוקול, ליד האצטרובלים הזכריים, גם נבדק על שלבים שונים של צמח יורה ועל שניהם עוברים zygotic ו סומטיים מ פ אשוח עם תוצאות טובות (תוצאות לא פורסם;. Karlgren ואח&#39;).

<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

תפוקה גבוהה ניתוח טכנולוגיות ביטוי הזמינים כיום לתת למדענים הצפת פרופילים הביטוי אך ההחלטה שלהם במונחים של הביטוי רקמות ספציפיות מוגבל בגלל בעיות לנתח רקמות הפרט. נתונים הביטוי צריך להיות אישר השלימו עם דפוסי ביטוי באמצעות למשל הכלאה באתרו, טכניקה המשמשת למקם mRNA התא ביטוי ספציפי. בשיטת הכלאה באתרו הוא מייגע, זמן רב ולעיתים קרובות דורש אופטימיזציה נרחב בהתאם מינים ורקמות. בניסויים באתרו יחסית קשה יותר לבצע מינים עציים, כגון נורווגיה כוניפאר אשוחית (Picea אשוח). כאן אנו מציגים DIG שונה בפרוטוקול הכלאה באתרו, אשר הוא מהיר החלים על מגוון רחב של מיני צמחים כולל פ אשוח. עם רק כמה התאמות, כולל שינה RNase טיפול וריכוז proteinase K, אנחנו יכולים להשתמש בפרוטוקול ללמוד ביטוי רקמות ספציפיות של גנים הומולוגיים איברי הרבייה הגברית gymnosperm אחד ושני מינים angiosperm: פ אשוח, thaliana ארבידופסיס ו Brassica napus. פרוטוקול עבדו באותה מידה עבור מינים גנים למד. AtAP3 ו BnAP3 נצפו השני והשלישי איברים פרחים במערבולת א &#39; thaliana ו-B napus ו DAL13 ב microsporophylls של האצטרובלים הזכריים מ פ אשוח. עבור פ אשוח K proteinase ריכוז, המשמש permeablize הרקמות, היה צריך להיות גדל ל 3 גרם / מ&quot;ל במקום 1 גרם / מ&quot;ל, ככל הנראה בשל רקמות יותר קומפקטי רמות גבוהות יותר של phenolics ופוליסכרידים. עבור כל המינים הטיפול RNase הוסר בשל עוצמת האות מופחת ללא גידול מקביל סגוליות. על ידי השוואת ספציפיים רקמות ביטוי דפוסים של גנים הומולוגיים משני הצמחים ועץ מחטניים אנו מראים כי ה-DIG בפרוטוקול באתרו שהוצגו כאן, רק עם שינויים מזעריים, ניתן ליישם במגוון רחב של מיני צמחים. לפיכך, פרוטוקול נמנע גם אופטימיזציה של מינים ספציפיים נרחב ושימוש מייגע של בדיקות שכותרתו רדיואקטיבית לטובת בדיקות DIG שכותרתו. בחרנו להדגים את השלבים טכנית בדרישה של פרוטוקול בסרט שלנו. אנה Karlgren וג&#39;ני Carlsson תרמו באופן שווה על מחקר זה. המחברים מקבילים: אנה Karlgren ב Anna.Karlgren @ ebc.uu.se ו Jens Sundström פ ב Jens.Sundstrom @ vbsg.slu.se

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

אנו מתארים DIG שונה<em&gt; באתרו</em&gt; פרוטוקול הכלאה, אשר הוא מהיר החלים על מגוון רחב של מיני צמחים כולל נורבגיה אשוח. עם רק כמה התאמות, כולל שינה RNase טיפול וריכוז proteinase K, הפרוטוקול ניתן להשתמש במחקרים של רקמות מינים שונים.

Non-רדיואקטיבי באתרו הכלאה פרוטוקול ישים עבור נורווגיה Spruce ומגוון של מיני צמחים

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

20.9K Views.  Uppsala University. אנו מתארים DIG שונה באתרו פרוטוקול הכלאה, אשר הוא מהיר החלים על מגוון רחב של מיני צמחים כולל נורבגיה אשוח. עם רק כמה התאמות, כולל שינה RNase טיפול וריכוז proteinase K, הפרוטוקול ניתן להשתמש במחקרים של רקמות מינים שונים.

Video: 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.

פעמיים פלואורסצנטי באתרו הכלאה בסעיפים המוח טריים

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

Neuroscience

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.

בחיי עיר טכניקות הכלאה פרפין-מוטבע דגימות אלמוגים למבוגרים

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 ...

Environment

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.

פלואורסצנציה<em&gt; באתר</em&gt; הכלאות (דגים) ללוקליזציה של וירוסים וחיידקים Endosymbiotic בצמח ורקמות חרקים

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

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.

Wholemount ב באתרו הכלאה עבור עוברי Astyanax

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

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.

איתור של הגנום והתמלילים של וירוס ה-DNA מתמיד ברקמות עצבית על ידי פלורסנט באתרו הכלאת שילוב עם 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 ...

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.

שילוב קרינה זוגית<em&gt; באתרו</em&gt; כלאה עם Immunolabelling עבור איתור של הביטוי של שלושה גנים בסעיפי מוח עכבר

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.

ב הכלאה באתרו עבור לוקליזציה המדויק של תמלילי בצמחים

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

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.

רדיואקטיבי באתרו הכלאה לזיהוי תבניות ביטוי מגוונות גנים רקמות

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).

-Cut ההיברידי: שיטת חתך משופרת עבור דוגמאות רקמות צמח סרבן

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.

Non-רדיואקטיבי באתרו הכלאה פרוטוקול ישים עבור נורווגיה Spruce ומגוון של מיני צמחים

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