The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.

הערה: תמיד להיות בטוח שאישור ועדת IACUC או אתיקה הוא במקום לפני תחילת כל הליך ניסיוני באמצעות דג הזברה. 1. הכינו חומרים כימיים והתקנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את הפתרונות הבאים. מצא את המתכונים לכל הפתרונות הללו במדריכי דג הזברה סטנדרטיים 10. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 מיליליטר של מים ביצה </li><li style=";text-align:right;direction:rtl"> 200 מיליליטר של 0.03% Tricaine בביצת מים </li><li style=";text-align:right;direction:rtl"> של נאגר מלוח פוספט 1x 100 מיליליטר (PBS) </li><li style=";text-align:right;direction:rtl"> RBC תמוגה מאגר (אופציונאלי) </li><li style=";text-align:right;direction:rtl"> חיץ יעד של בחירה (מקבע, Trizol, PBS, וכו &#39;) בצינור 1.5 מיליליטר microfuge על קרח הערה: מקבע וTrizol רעילים אם הם באים במגע עם עור. ללבוש כפפות בעת טיפול בחומרים אלה. </li></ul></li><li style=";text-align:right;direction:rtl"> הכן התקנה לנתיחה: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מקם את מקור מתכווננת אור על מיקרוסקופ לנתח. מניחים את המכסה של 9 סנטימטר צלחת פטרי על הבמה מיקרוסקופ לנתח, ויוצקים שכבה דקה של 1x PBS לזה (המכסה משמש כי המנה עצמה היא עמוקה מדי עבור נתיחה קלה). </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת פטרי עצמו (לא את המכסה) על הדלפק ליד ההיקף לנתח ויוצקים כ 15 מיליליטר של 1x PBS בזה. להשתמש בזה כדי לשטוף את הלב לאחר נתיחה. לחלופין, להשתמש RBC תמוגה הצפת. </li><li style=";text-align:right;direction:rtl"> להב מקום גילוח, שני זוגות מלקחיים, microscissors (אופציונאלי), ופיפטה העברה לצד מיקרוסקופ. להשיג מיכל של קרח אם המתת חסד על ידי קירור מהיר נבחרה. </li><li style=";text-align:right;direction:rtl"> יוצקים 200 מיליליטר של 0.03% Tricaine בביצת מים לתוך כוס זכוכית 250 מיליליטר אם המתת חסד על ידי Tricaine נבחר. הכן תיק Biohazard קטן לסילוק פגרי דג הזברה. </li></ol></li></ol> 2. הכן דג הזברה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדגים קבועים, להביא דגים קבועים, לשטוף בPBS, להתקנת מיקרוסקופ. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את כל דג הזברה מבוגרת על ידי קירור מהיר עם קרח ל&gt; 10 דקות. </li><li style=";text-align:right;direction:rtl"> שימוש במלקחיים כדי לעשות חור בעור מעל הצפק(מהלב) כדי לסייע לחדירה מקבע, ואז לשים אותם בparaformaldehyde 4% בתקן Buffer 10 לשעה 2 ב RT או O / N ב 4 ° C. </li></ol></li><li style=";text-align:right;direction:rtl"> לחלופין, לדגים טריים, דגי המקום להיות מורדמים במכל קטן החזקה ולהביא להתקנת מיקרוסקופ. בהתאם לפרוטוקול הדגים של המתקן הבודד והשימושים במורד הזרם ללב דגים גזורים, להרדים דגים עם Tricaine או להרדים על ידי קירור מהיר לפני עריפת הראש. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להשתמש בקרח, להרים דג אחד עם רשת הדגים והמקום במי קרח עד שהדג מפסיק לנוע, כ -5 דקות. </li><li style=";text-align:right;direction:rtl"> לחלופין, להשתמש Tricaine, להרים דג אחד עם רשת הדגים ומניחים בכוס של הפתרון עד תנועות זימים להפסיק. </li></ol></li><li style=";text-align:right;direction:rtl"> במהירות להרים את הדגים מורדמים על ידי סנפיר הזנב שלה ולהניח אותה בצד שלה בכיסוי צלחת פטרי שהיה מלא ב1x PBS. השתמש במלקחיים כדי להרים את סנפיר חזה ביד אחת, תוך השימוש בrazoלהב r לערוף את הדגים ביד השנייה, רק אחורי לצירופה של סנפיר החזה (איור 1 א). לבצע שלב זה או על ידי מחפש דרך מיקרוסקופ או פשוט ישירות לדמיין את הדגים על הבמה מיקרוסקופ. הערה: דגים טריים עדיין מורדמים ידממו לאחר עריפת הראש. </li></ol><img alt="איור 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> איור 1. לנתיחה לב מבוגר דג הזברה מנצלת ציוני דרך אנטומי דג הזברה. (א) כדי לערוף את הדגים, להרים את סנפיר החזה עם מלקחיים ולהשתמש בסכין גילוח נקי חד לאורך הקו המקווקו האדום כפי שמוצג. (ב) כדי לייצב את ראש הדג, מקום שן אחת של המלקחיים בפה הדג ואילו שן האחר טמון פני העין, ולאחר מכן להפעיל את ראש הדג, כך שמשטח הגחון הוא למעלה והן השיניים של המלקחיים יציבות נגד תחתית צלחת פטרי. (C) השתמש בforc החופשיeps לחתוך את הקובץ המצורף של operculum (החץ). (ד) ההרמה זו, אב העורקים הגב גלוי כמבנה לבן עם פס ורוד המציין דם luminal (חץ). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> 3. לנתח הלב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דמיין את ראש הדג מתחת למיקרוסקופ. עם אחד מלקחיים, לייצב את ראש הדג על ידי הנחת שן אחת של המלקחיים בפה הדגים, דרך המוח, ואילו שן האחר הוא מחוץ לראש, על פני העין (איור 1). ודא שני השיניים של המלקחיים הן יציבים נגד תחתית צלחת פטרי. מחזיק את ראש הדג בדרך זו, משטח הגחון שלה צריך להיות כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> ביד השנייה, להשתמש במלקחיים השני לחתוך את הקובץ המצורף הגחון של operculum לגוף (חץ, איור 1 ג). ההרמה מעט זה עם המלקחיים, דאב העורקים orsal יופיעו מתחת למבנה לבן עם פס אדום של דם בלומן של אב העורקים (חץ, 1D איור). חותך את אב העורקים גב עם המלקחיים על ידי צובט את אב העורקים ומושכים כלפי מעלה; לחלופין, להשתמש microscissors לחתוך את אב העורקים הגב. </li><li style=";text-align:right;direction:rtl"> עכשיו, להשתמש במלקחיים השני לתפוס את סנפיר החזה מבסיסו, ובכלל זה סחוס הגוף בבסיס של הסנפיר, ולהרים את זה ממני. חזור עם סנפיר החזה האחר. מדי פעם, הלב יוצא עם סנפירי החזה, כך לבחון חלקים אלה כדי לוודא שהלב אינו מחובר לפני השלכת אותם. הערה: בשלב זה, הלב צריך להיות גלוי, עדיין שלם ומחוברים לראש הדג שנותר (אם כי מדי פעם זה יורד עם סנפירי החזה). הלב יכול להיות מזוהה על ידי צורתו, צבעו הוורוד, והיותו מוקפים בקרום לב פיגמנט. משום המתת חסד של הדגים מתבצע במהירות, לב דגים טרי מורדמים עדיין עשוי להיות מכות slowly. </li><li style=";text-align:right;direction:rtl"> עזוב את ראש הדג עם המלקחיים הראשונים, כך ששני הידיים מחזיקות מלקחיים וחופשיים. להשתמש בשני המלקחיים כדי להקניט את הלב מקרום הלב. לתפוס את הלב על ידי השארית של אב העורקים הגב תוך קרום הלב הנותר התרחק. הערה: אם קבוע או טרי, הלב הוא חזק כדי עדין מושך תוך רקמת חיבור המקיפה היא פריך יותר. לכן, כל רקמת קרום הלב שמסביב יכולה להיות גזור משם עם הלב שנותר ללא פגע. </li><li style=";text-align:right;direction:rtl"> מחק את פגר דגי שאריות בשקית Biohazard. </li></ol> 4. מכין את הלב ליישומים במורד הזרם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים, לתפוס את הלב על ידי הקצה של אב העורקים גב / arteriosus bulbus ולמקם את הלב בצלחת הטרי של 1x PBS. לחלופין, להשתמש פיפטה העברה. הזז את הלב הלוך ושוב בPBS על 10x כדי לשטוף כמה שיותר דם ככל האפשר. </li><li style=";text-align:right;direction:rtl"> עבור יישומים בהם הוא חשוב כדי להסיר את כל possibתאי דם le, לשטוף בצלחת פטרי 9 סנטימטר עם 15 מיליליטר RBC תמוגה הצפת במקום PBS, תוך שימוש באותה תנועת גב ו-ושוב. אם השמירה על מבנה הלב אינה חשובה ליישום במורד הזרם (לדוגמא, מה שהופך את RNA), להשתמש במלקחיים או microscissors כדי לפתוח את חלל הלב ולהקל על השטיפה במרחק של תאי דם. </li><li style=";text-align:right;direction:rtl"> עבור יישומים בי חדרי לב נפרדים רצויים, להשתמש במלקחיים או microscissors לנתח את arteriosus bulbus, אטריום, והחדר מלבד אחד את השני. </li><li style=";text-align:right;direction:rtl"> העבר את הלב, או תאים נפרדים, למאגר שהיעד על קרח. הערה: יעדים נפוצים כוללים מאגרים לתא דיסוציאציה, paraformaldehyde 4%, או Trizol. </li></ol>

<ol>
	<li>Arnaout, R., 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=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., 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=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., 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=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., 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=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

The authors have no disclosures.

בעוד שיטות ללנתח לב דג הזברה המבוגר תוארו, שיטות אלה היו זמן רב ונפוצות גרמו לפגיעה בלב במהלך נתיחה. כדי לבצע ניסויים שבם מספר רב של לבבות מבוגרים עשוי להיות נחוץ, ו / או כאשר הימנעות השפלה של רקמת לב היא חשובה עבור יישומים במורד הזרם, הזמן הנדרש תוך שימוש בטכניקות מסור?...

Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >small tank for transporting fish</td>
			<td >Aquaneering</td>
			<td >ZHCT100</td>
			<td></td>
		</tr>
		<tr >
			<td >fish net</td>
			<td >Petsmart</td>
			<td >36-16731</td>
			<td></td>
		</tr>
		<tr >
			<td >250mL glass beaker</td>
			<td >Kimble</td>
			<td >14005-250</td>
			<td></td>
		</tr>
		<tr >
			<td >9cm polystyrene petri dish</td>
			<td >Nunc</td>
			<td >172958</td>
			<td></td>
		</tr>
		<tr >
			<td >razor blade</td>
			<td >Personna American Safety Razor Company</td>
			<td >94-120-71</td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont #5SF forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-00</td>
			<td></td>
		</tr>
		<tr >
			<td >dissecting microscope</td>
			<td >Olympus</td>
			<td >SZX16</td>
			<td></td>
		</tr>
		<tr >
			<td >Tricaine</td>
			<td >Sigma</td>
			<td >A-5040</td>
			<td></td>
		</tr>
		<tr >
			<td >plastic transfer pipette</td>
			<td >Thermo Scientific</td>
			<td >202-20S</td>
			<td></td>
		</tr>
		<tr >
			<td >gooseneck light source</td>
			<td >Dolan-Jenner Industries, Inc</td>
			<td >Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td>
			<td></td>
		</tr>
		<tr >
			<td >fluorescent light source</td>
			<td >Lumen Dynamics</td>
			<td >X-Cite 120Q</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >micro-scissors</td>
			<td >Biomedical Research Instruments, Inc</td>
			<td >11-1000</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >RBC Lysis Buffer</td>
			<td >eBioscience</td>
			<td >00-4333-57</td>
			<td>optional</td>
		</tr>
	</tbody>
</table>

recovery of adult zebrafish hearts for high-throughput applications

שימוש במערכת מודל דג הזברה ללימוד פיתוח, התחדשות, ומחלה מתפשטת לכיוון שימוש בלבבות מבוגרים לניתוק תא וטיהור של RNA, DNA, חלבונים ו. כל היישומים האלה דורשים התאוששות המהירה של מספר המשמעותי של לבבות דג הזברה להימנע גן בקרה, חילוף חומרים, ושינויים אחרים שמתחילים אחרי המוות. לב דג הזברה מבוגר נדרש גם ללימוד מבנה לב עבור מגוון רחב של מוטציות וללימוד התחדשות לב. עם זאת, לנתיחה לב דג הזברה המסורתית היא איטית וקשה ודורשת כלים מיוחדים, מה שהופך את הנתיחה בקנה מידה גדולה של לבבות דג הזברה מבוגרים מייגע. שיטות מסורתיות גם נמל את הסיכון של פגיעה בלב במהלך הניתוח. כאן, אנו מתארים שיטה לניתוח לב דג הזברה מבוגר שהיא מהיר, לשחזור, ומשמר את ארכיטקטורת לב. יתר על כן, שיטה זו אינה דורשת כלים מיוחדים, אינו מכאיב לדג הזברה,יכול להתבצע על דגימות טריות או קבועים, וניתן לבצעו בדג זברה צעירה כמו בן חודש. הגישה המתוארת מרחיבה את השימוש בדג הזברה מבוגרת למחקר לב וכלי דם.

באמצעות שיטה זו, לב דג הזברה מבוגר יכול להיות גזור בדקות פחות מ 1, בהשוואה ליותר מ -5 דקות בשיטות מסורתיות 8. לבבות גזורים בשיטה זו הם באופן מהימן שלמים (איור 2 א), ואילו שיטות מסורתיות 8 דורשות חיתוך בצורה עיוורת לקרום הלב ולכן בדרך כלל לגרום לניזק או ?...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

שחזור של לבבות למבוגרים דג הזברה עבור יישומי תפוקה גבוהה

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Research

JoVE Journal

Biology

12.2K Views.  University of California San Francisco. Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Video: Recovery of Adult Zebrafish Hearts for High-throughput Applications

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

שיטת תפוקה גבוהה עבור cryopreservation זרע דג הזברה במבחנה הפריה

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

הזרקה לתוך intraperitoneal דג הזברה למבוגרים

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

תפוקה גבוהה Saccharification Assay חומרים Lignocellulosic

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

ב electroporation vivo של Morpholinos לתוך הרשתית דג הזברה למבוגרים

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

ב electroporation vivo של Morpholinos לתוך למבוגרים התחדשות זנב סנפיר דג הזברה

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

הדם אוסף של ניתוח ביוכימי דג הזברה למבוגרים בלבד

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging דג הזברה למבוגר

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

מסך ידני מולקולה קטנה מתקרב תפוקה גבוהה באמצעות עוברי דג הזברה

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

איסוף דם חוזרים ונשנים לבדיקות דם בדג הזברה למבוגרים

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...