עבודה זו נתמכה על ידי Generalitat דה קטלוניה (2005-SGR00437). המחברים מודים ל י פ ו בלאנקו וידאל לפיקוח שלהם עידוד, ג&#39;לוקאס לקבלת סיוע טכני שלו.

I. עיבוד המדגם קיבוע התא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השאירו את מדגם הזרע במכלים סטריליים בטמפרטורת החדר למשך 20 דקות עד עיבוי. </li><li style=";text-align:right;direction:rtl"> מעבירים את המדגם כדי צינור צנטריפוגות ספין אותו 1000g במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הוצא בעדינות וזורקים supernatant באמצעות פיפטה פסטר. </li><li style=";text-align:right;direction:rtl"> הוסף פתרון hypotonic (KCl, 0.075M) מחומם מראש על 37 ° C, טיפה אחר טיפה, תוך כדי ערבוב על מערבולת לקבל נפח סופי של 10 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> מניחים את הצינור באמבטיה המים 37 מעלות צלזיוס במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 1000g במשך 5 דקות. בזהירות להתעלמות supernatant ידי יציקה, מבלי להפריע גלולה. </li><li style=";text-align:right;direction:rtl"> לאחר resuspending גלולה, להוסיף המוכן טרי (מתנול 03:01: חומצה אצטית) מקבע של Carnoy טיפה אחר טיפה תוך כדי ערבוב על מערבולת לקבל נפח הסופי של 8 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> חזור נקודות 6 ו -7 פעמים רבות ככל הדרוש כדי להשיג גלולה לבנה. </li><li style=";text-align:right;direction:rtl"> הוסף מתנול מוכן טרי: חומצה אצטית (03:01) טיפה אחר טיפה כדי לכוונן את עוצמת הריכוז הסופי הסלולר הנדרש לקבלת תא טובה מתפשטת (באותם מקרים עם כדורית קטנה, כמה טיפות יספיקו ואילו דגימות עם כמות גבוהה הזרע התאוששה, הנפח הסופי יכול להגיע 1-2 מ&quot;ל). שקופית הבדיקה יכולה להתבצע על ידי הטלת המדגם resuspended לשקופית משומנת ובוחן אותו תחת מיקרוסקופ לעומת שלב. זה יאפשר לבדוק את פיזור הסלולר שהושג יאפשר תיקון זה, אם יש צורך, על שקופיות נוספות. </li><li style=";text-align:right;direction:rtl"> כדי להפוך את הרחבות, ירידה מגובה מינימלי של 40 ס&quot;מ שתיים או שלוש טיפות של השעיה תא למרכז שקופיות unfrosted (מאוחסן בעבר ב -20 מעלות צלזיוס מתנול כדי degrease אותם). כדי לאפשר את מיקומו של המדגם לאורך הפרוטוקול, לסמן את האזור המכיל את הזרע הרחבה בצד השני של השקופית באמצעות עיפרון היהלומים. </li><li style=";text-align:right;direction:rtl"> אחסן את השקופיות ב -20 מעלות צלזיוס למשך 24 שעות לפחות. הערה: תוספת של מתנול: חומצה אצטית (03:01) טיפה אחר טיפה תוך כדי ערבוב היא צעד חשוב מאוד כדי למנוע היווצרות של אגרגטים הזרע. </li></ol> השנייה. Decondensation <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השקופיות המאוחסן -20 ° C חייב להיות מופשר להמשיך עם פרוטוקול (להשאיר אותם בטמפרטורת החדר). </li><li style=";text-align:right;direction:rtl"> מניחים את השקופית בשתי צנצנות coplin רצופים עם תמיסת מלח נתרן 2x ציטרט (2xSSC) למשך 3 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> העברת השקופית באמצעות סדרה של אתנול שוטף למשך 2 דקות בתוך צנצנת כל coplin. התחל עם 70% אתנול, פעל על ידי 90% ולסיים עם 100%. לייבש את השקופית להשאיר אותו בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> דגירה השקופית בתמיסה dithiothreitol (1,4-dithiothreitol 5mm, טריטון 1% X-100, 2-אמינו-2 (hydroxymethyl) -1,3-propanediol 50mm) בשעה 37 ° C בחממה כדי decondense הכרומטין. מוצר זה פועל על ידי שבירת גשרים דיסולפיד של protamines כי סליל ה-DNA בגרעין תא הזרע. זהו צעד חשוב מאוד כי ה-DNA בגרעין הזרע נדחס מאוד. זמן הדגירה של שקופיות פתרון dithiothreitol (DTT) חייב להיות מותאם על פי התגובה של מדגם למוצר זה. בדרך כלל, הפעם הוא סביב 8 דקות, למרות שזה יכול להשתנות 2-15 דקות. </li><li style=";text-align:right;direction:rtl"> מיד להעביר את השקופית שתי צנצנות ברציפות coplin עם 2xSSC במשך 3 דקות coplin כל אחד. </li><li style=";text-align:right;direction:rtl"> המשך על ידי הצבת את השקופית באמצעות סדרה של אתנול שוטף למשך 2 דקות בתוך צנצנת כל coplin (70%, 90% ו 100% אתנול). תנו את השקופית להתייבש בטמפרטורת החדר. הערה: חשיפת יתר DTT תביא לפיזור אותות FISH בסוף ההליך, ואילו חשיפה קצר מדי יגרום לחוסר אותות מסוימים. </li></ol> ג. הכלאה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפגל את ה-DNA של הזרע דוגרים השקופית בצנצנת coplin עם פתרון לפוראמיד (70% Formamide/2xSSC) בשעה 73 ° C במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> העברת השקופית באמצעות פתרונות אתנול דקה 1 לכל coplin קנקן (להתחיל עם אתנול 70%, 85% ולסיים עם 100%). השאירו את השקופית להתייבש בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הוסף ישיר l של 5 המקביל מוכן לשימוש תערובת בדיקה לחמוק כיסוי 15x15 (Assay AneuVysion Multi-Probe צבע פאנל: CEP 18/X/Y או LSI 13/21). </li><li style=";text-align:right;direction:rtl"> כפי שזה מוצג הוידאו, בזהירות במקום להחליק על פתק את המכסה כדי להרכיב את אזור היעד את התערובת בדיקה. חותם זה במלט גומי. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופית לתוך טרום חימם 37 ° C קאמרית הכלאה (HYBrite ™) ו דגירה אותו 37 מעלות צלזיוס למשך 6-24 שעות. הערה: בעוד denaturation המדגם הזרע היא חובה, את הצורך denaturing בדיקות נקבע על ידי היצרן. תערובות בדיקה השתמשו בפרוטוקול הזה מוכנים לשימוש, במקרה זה, denaturation בדיקה אינה נדרשת. נפח התערובת בדיקה ישתנה בהתאם לגודל האזור הכלאה. </li></ol> IV. רוחצתלאחר הכלאה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח את השקופית מהאולם הכלאה. </li><li style=";text-align:right;direction:rtl"> הסר את המלט גומי בזהירות לשלוף את הפתק מכסה מחליק בעדינות הצידה. </li><li style=";text-align:right;direction:rtl"> כדי למנוע את האותות הכלאה נוקבים במקום להחליק במשך 2 דקות בתוך צנצנת עם coplin% 0.4xSSC/0.3 NP-40 מראש התחמם ב 73 מעלות צלזיוס באמבט מים. </li><li style=";text-align:right;direction:rtl"> העברת שקופיות פתרון NP-40 לשטוף 2xSSC/0.1% בטמפרטורת החדר למשך דקה 1. תנו את השקופית להתייבש בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הוסף מוצר counterstaining לאזור היעד (8 l עבור להחליק לכסות 18x18). המוצר הנפוץ ביותר הוא 4 &#39;,6-diamidino-2-phenylindole (DAPI, Vysis Inc). </li><li style=";text-align:right;direction:rtl"> כפי שזה מוצג הוידאו, לכסות את אזור הכלאה עם תלוש כיסוי (כדי לשמר את ההכלאה להחליק לכסות יכול להיות חתום עם לכה מסמר). </li><li style=";text-align:right;direction:rtl"> חנות שקופיות הכלאה ב -20 ° C בחושך עד ניתוח הסיכוי שלהם. בתנאים אלה השקופיות ניתן לאחסן עד 12 חודשים ללא משמעותי לאיבוד עוצמת האות פלואורסצנטי. הערה: הטמפרטורה גבוהה או זמן לשטוף מופרז עלול לגרום לחיסול של אותות מסוימים, ואילו השני לא היה לשטוף את הכלאות בדיקה נוקבים. היקף המוצר counterstaining הוסיף ישתנה בהתאם לגודל האזור הכלאה לכסות. </li></ol> V. ויזואליזציה ההכנות ניתן להעריך תחת מיקרוסקופ epifluorescence מצויד במסנן הלהקה משולשת עבור DAPI / טקסס אדום / FITC ו יחיד הלהקה מסננים עבור אקווה, FITC וטקסס האדום. קריטריונים להערכה סטנדרטית חייב להיות במעקב במשך הערכה נכונה של גרעיני זרע 1. הכנת הכלאה עם בדיקות של כרומוזומי X, Y ו 18 צריכים להציג אותות עבור שלושת הכרומוזומים. כל זרע נורמלית חייב להראות איתות כחול (המקביל ל כרומוזום 18), ואת אות ירוק (X נושאות זרע) או איתות אדום (Y נושאות זרע). בהכנת הכלאה עם בדיקות של כרומוזומים 13 ו - 21, אות ירוק כרומוזום 13 ו סימן אדום על כרומוזום 21 יש להבחין בכל זרע נורמלית.

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

פרוטוקול זה מתאר כיצד תהליך דגימות זרע אנושי להשגת FISH ההכנות הזרע. באמצעות פרוטוקול זה ניתן לנתח פגמים בכרומוזומים של גמטות זכריות. ההקרנה זרע aneuploidy יש יישומים גם בהקשר של מחקר בסיסי בייעוץ הרבייה שניתנה זכרים עקרים. למרות האבחנה הקלינית הכרומוזומים ביותר למד נרחב ה...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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fluorescence in situ hybridization (fish) protocol in human sperm

Aneuploidies הם פגמים בכרומוזומים השכיח ביותר בבני אדם. רוב הפרעות אלו נובעות שגיאות meiotic בתהליך gametogenic אצל ההורים. אצל גברים אנושיים, שגיאות אלה יכולים להוביל לייצור של זרע עם הפרעות כרומוזום מספריים המייצגים סיכון מוגבר להעברת אנומליות אלה לצאצאים. מסיבה זו, את הטכניקה של הקרינה הכלאה באתרו (FISH) על גרעין הזרע הפך פרוטוקול שולבו נרחב בהקשר של אבחנה קלינית. בפועל זה מספק אומדן של תדרים של הפרעות כרומוזום מספריים הגמטות של המטופלים המבקשים ייעוץ גנטי הרבייה. עד כה, את הכרומוזומים כללו לרוב בניתוח FISH הזרע הם כרומוזומי X, Y, 13, 18 ו - 21. זה וידאו מאמר זה מתאר, צעד אחר צעד, איך לעבד ולתקן דגימת זרע אנושי, איך decondense ו לפגל הכרומטין זרע, כיצד להמשיך לקבל ההכנות FISH הזרע, וכיצד לחזות את התוצאות במיקרוסקופ. הערות מיוחדות הצעדים המשמעותיים ביותר ניתנים על מנת להשיג את התוצאות הטובות ביותר.

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Fluorescence in situ hybridization FISH Protocol in Human Sperm

זה וידאו מאמר זה מתאר, צעד אחר צעד, כיצד תהליך דגימת זרע כדי להשיג באיכות טובה פלואורסצנטי<em&gt; באתרו</em&gt; הכלאה על בזרע אנושי. ההכנות שהושגו ניתן להשתמש להקרנה aneuploidy בהקשר של אבחנה קלינית.

פלואורסצנציה באתרו הכלאה (FISH) פרוטוקול של זרע אדם

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K Views.  Universitat Autónoma de Barcelona. זה וידאו מאמר זה מתאר, צעד אחר צעד, כיצד תהליך דגימת זרע כדי להשיג באיכות טובה פלואורסצנטי באתרו הכלאה על בזרע אנושי. ההכנות שהושגו ניתן להשתמש להקרנה aneuploidy בהקשר של אבחנה קלינית.

Video: Fluorescence in situ hybridization FISH Protocol in Human Sperm

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

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

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

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

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

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

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

זיהוי של רנ&quot;א נגיפי על ידי הקרינה<em&gt; באתרו</em&gt; הכלאה (דגים)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

ניתוח של שעתוק גנים מהתא הבודד על ידי RNA פלורסנט<em&gt; באתר</em&gt; הכלאה (דגים)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

עיתוי מעתיק כרומוזום בשילוב עם פלורסנט<em&gt; באתר</em&gt; הכלאה

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

הדמיה וניתוח של מולקולות ה-mRNA באמצעות הקרינה<em&gt; באתרו</em&gt; הכלאה ב<em&gt; Cerevisiae Saccharomyces</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; באתרו</em&gt; הכלאה לרקמות כליה פורמלין קבועה

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

DNA-RNA בשילוב פלורסנט<em&gt; באתרו</em&gt; הכלאה (דגים) לחקר איון כרומוזום X בתאי גזע הבדיל נקבה עכבר עוברי

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

המהיר פלורסנט<em&gt; באתר</em&gt; הכלאת פרוטוקול לXist RNA משולב עם Immunofluorescence של היסטון שינוי באיון כרומוזום X

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

שיטה פשוטה DNA הקרינה<em&gt; באתר</em&gt; הכלאה לכרומוזומים &quot;סתמת

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

תצפית כימות הטלומרים ו רצפים חוזרים קרינה באמצעות<em&gt; באתרו</em&gt; הכלאה (דגים) עם PNA בדיקות ב<em&gt; Elegans Caenorhabditis</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...