אנו אסירי תודה לרות ליהמן וDorthea Godt שעושים חסד סיפק נוגדנים ושה ופקק התנועה. ברצוננו להודות למרכז מניות Bloomington באוניברסיטת אינדיאנה לשמירה על מניות ובלבד ובנק Hybridoma מחקרים התפתחותיים שפותח בחסות NICHD ומתוחזק על ידי אוניברסיטת איווה. אנו מודים לכל החברים במעבדה Wawersik לייעוץ והתמיכה שלהם. עבודה זו מומנה על ידי מונרו חוקרי גרנט התכנית (לAF וLB) וNSF להעניק IOS0823151 (לMW).

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C., Bat, M., Martinez Arias, A. <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 Development of Drosophila melanogaster. , 1-70 (1993).</li><li>Eliazer, S., Buszczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+a+niche:+studies+from+the+Drosophila+ovary.">Finding a niche: studies from the Drosophila ovary.</a> Stem Cell Res Ther. 2, 45 (2011).</li><li>Sahut-Barnola, I., Godt, D., Laski, F. A., Couderc, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+ovary+morphogenesis:+Analysis+of+terminal+filament+formation+and+identification+of+a+gene+required+for+this+process.">Drosophila ovary morphogenesis: Analysis of terminal filament formation and identification of a gene required for this process.</a> Developmental Biology. 170, 127-135 (1995).</li><li>Godt, D., Laski, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+rearrangement+and+cell+recruitment+in+Drosophila+ovary+morphogenesis+and+the+requirement+of+bric+a+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric a brac.</a> Development. 121, 173-187 (1995).</li><li>Gancz, D., Lengil, T., Gilboa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+regulation+of+niche+and+stem+cell+precursors+by+hormonal+signaling.">Coordinated regulation of niche and stem cell precursors by hormonal signaling.</a> PLoS Biol. 9, e1001202 (2011).</li><li>Matsuoka, S., Hiromi, Y., Asaoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egfr+signaling+controls+the+size+of+the+stem+cell+precursor+pool+in+the+Drosophila+ovary.">Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary.</a> Mech Dev. 130, 241-253 (2013).</li><li>Song, X., Zhu, C. H., Doan, C., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296, 1855-1857 (2002).</li><li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila.+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila. neuroblasts: a model for stem cell biology.</a> Development. 139, 4297-4310 (2012).</li><li>Urbach, R., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblast+formation+and+patterning+during+early+brain+development+in+Drosophila.">Neuroblast formation and patterning during early brain development in Drosophila.</a> BioEssays. 26, 739-751 (2004).</li><li>Bello, B., Reichert, H., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+gene+negatively+regulates+neural+progenitor+cell+proliferation+in+the+larval+central+brain+of+Drosophila.">The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila.</a> Development. 133, 2639-2648 (2006).</li><li>Lee, C. Y., Wilkinson, B. D., Siegrist, S. E., Wharton, R. P., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brat+is+a+Miranda+cargo+protein+that+promotes+neuronal+differentiation+and+inhibits+neuroblast+self-renewal.">Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.</a> Dev Cell. 10, 441-449 (2006).</li><li>Betschinger, J., Mechtler, K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+segregation+of+the+tumor+suppressor+brat+regulates+self-renewal+in+Drosophila+neural+stem+cells.">Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.</a> Cell. 124, 1241-1253 (2006).</li><li>Pereanu, W., Shy, D., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+proliferation+of+the+larval+brain+glia+in+Drosophila.">Morphogenesis and proliferation of the larval brain glia in Drosophila.</a> Dev Biol. 283, 191-203 (2005).</li><li>Moraru, M. M., Egger, B., Bao, D. B., Sprecher, S. 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The authors have nothing to disclose.

פרוטוקול זה מספק שיטה להצלחת immunostain למקד דרוזופילה עוברית ורקמות זחל באתר, ובכך מבטל את הצורך בנתיחה. לפי פרוטוקולים לפני מכתים עוברים המוקדמים 1,2,3, קרום כוריוני מוסר באמצעות אקונומיקה 50% (NaOCl). דגימות קבועות בפורמלין ומתנול. בגלל ציפורן הזחל גורמת מדגם...

עוברי דרוזופילה וזחלים מספקים מודל מצוין ללמוד תהליכים התפתחותיים באיברים ורקמות רבים. הדמיה של תאים בודדים היא לעתים קרובות יש צורך במחקרים אלה על מנת לוודא הסביבות המורכבות שבו תאים לפתח. ויזואליזציה של תאים ברקמות יכולה להיות מושג באמצעות immunostaining. ובכן תיאר immunostaining פרוטוקולים קיימים לרקמות עוברי דרוזופילה &lt;17 שעות לאחר הטלת ביצים (AEL) 1-3. עם זאת, צורות מגן ציפורן לקראת הסוף של עובר, מניעת חלחול נוגדן יעיל. לפיכך, פרוטוקולי immunostaining אלה אינם יעילים בניתוח של רקמות בעוברים בשלב מאוחר ובשלבים מאוחרים יותר של התפתחות זחל (instar 1 st (L1), instar 2 nd (L2), ו3 instar rd (L3)). חוסר יעילות זה מטיל מחסום להבנה של תהליכים דינמיים המתרחשים בתקופה התפתחותית מורחבת זה 4. Tissנתיחת ue היא טכניקה מועסק נרחב לעקוף מכשול זה 5-7. עם זאת, לנתיחה יכולה להוכיח לא יעילה. חילוץ ניתן לשעבוד על ידי קושי באיתור או בידוד עוברי ורקמות זחל. יתר על כן, ההסרה הפיסית של רקמות היעד עלולה לגרום נזק על ידי פקיעתם או בכך שלא לחלץ אותם בשלמותם. Sonication הוא שיטה שמעסיקה גלי קול להפריע אינטראקציות מולקולאריים. זה כבר נעשה שימוש כדי לשבש את שלמות ציפורן זחל תסיסנית כדי immunostain פיתוח סוגי תאים עצביים 6. פרוטוקול זה הותאם לimmunostain עוברי בשלב מאוחר ובלוטות מין זחל, אשר יכול להיות קטנה כמו 50μm בקוטר 8-10. באמצעות מחקרים כאלה, התהליך של תאי גזע בתאי מין היווצרות נישה גברית (GSC) התאפיין בשלב מאוחר עוברי דרוזופילה 8-10 ומנגנוני ויסות התפתחות תאי גזע וDIFferentiation בבלוטות מין עוברי בשלב מאוחר וזחלים כבר הובהר 9-12. לפיכך, sonication מספק אלטרנטיבה יעילה לנתיחת רקמות שעלולות להיות קשה בגלל גודל של רקמה. יתר על כן, היא מאפשרת immunostaining של רקמות דרוזופילה באתר, עוזב תאים בהקשר של הגוף כולו ושמירה במורפולוגיה באתר. כאן, אנו מתארים פרוטוקול צעד אחר צעד לimmunostaining הקרינה של עוברי בשלב מאוחר עד תחילת / רקמות אמצע-L3 באתר. ניתוח של רקמת אשכים ועצביים דרוזופילה מוצג בנציג התוצאות כדי להדגים את היעילות של פרוטוקול זה. יתר על כן, פרוטוקול immunostaining זה עשוי להיות מותאם לנתח רקמות אחרות דרוזופילה, כמו גם רקמות באורגניזמים אחרים עם עור מת חיצוני.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1: Reagents and Buffers</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Triton X-100 (PBTx)</td>
			<td>For 5 L: 500 mL PBS 10X 4.45 L ddH2O 50 mL Triton 10%</td>
			<td>Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline 10x (PBS 10X)</td>
			<td>For 1 L in dH2O: 80 g NaCl 2 g KCl 14.4g Na2HPO4 2.4 g KH2PO4</td>
			<td>Add components and fill to appropriate volume. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton 10%</td>
			<td>For 50 mL: 5 mL of Triton 5 mL of PBS 10X 45 mL of ddH2O</td>
			<td>Rock to mix. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>PEMS</td>
			<td>0.1 M Pipes (pH 6.9) 2.0 mM MgSO4 1.0 mM EGTA</td>
			<td>Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>For a 400ml of a 0.25 M solution (pH 6.9): 30.24 g Pipes dH20 NaOH</td>
			<td>Dissolve Pipes in 300 mL dH2Oand then adjust to pH 6.9 with NaOH. Bring the total volume to 400 mL with dH2O and autoclave. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>37% formaldehyde by weight in methanol</td>
			<td>Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>n-Heptane</td>
			<td>CAS 142-82-5. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Methanol</td>
			<td>CAS 67-56-1. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Tween (PBTw)</td>
			<td>To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10%</td>
			<td>Filter sterilize after adding all components. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 10%</td>
			<td>For 50 mL: 5 mL Tween 5 mL of PBS 10X 40 mL of ddH2O</td>
			<td>Rock to mix. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin/Phosphate Buffer Tween (BBTw)</td>
			<td>To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% 1 g Bovine Serum Albumin (BSA)</td>
			<td>Add BSA then sterilize using a 0.2 micrometer vacuum filter unit. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>To make 10 mL: Normal Goat Serum (Jackson ImmunoResearch Laboratories Code: 005-000-121) 10 mL ddH2O</td>
			<td>Add ddH2O to vial of NGS and sterilize using a 0.2 micrometer syrninge filter. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-diazabicyclo[2.2.2]octane (DABCO)</td>
			<td>To make 100 mL: 25 mL ddH2O 1 mL Tris HCl (1M, pH 7.5) 2.5 g of DABCO solid (CAS: 281-57-9) 3.5 mL 6N HCl 250 uL 10N NaOH 70 mL glycerol</td>
			<td>In 250 mL beaker with stir bar, add ddH2O, Tris HCl and DABCO. Stir and then add 6N HCl, 10 N NaOH, and glycerol. Then add 10NaOH dropwise until solution reaches pH 7.5. Aliquot. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO + p-phenylenediamine (PPD) Solution</td>
			<td>1.765 mL NaHCO3 0.353 Na2CO3 0.02 g PPD (CAS: 106-50-3)</td>
			<td>Dissolve PPD in NaHCO3 and NaCO3 solution. Add 60 uL of PPD solution to 500 uL of DABCO. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple juice plates</td>
			<td>To make ~200 plates: 45 g agar (CAS#9002-18-0) 45 g granulated sugar (store bought) 500 mL Apple juice (store bought) 15 mL Tegosept 10% 1.5 mL ddH2O</td>
			<td>Add agar to ddH2O in 4L flask then autoclave for 30 minuntes. Mix apple juice and sugar on heated stir plate. Gradually add apple juice mixture to autoclaved agar. Mix on heated stir plate then aliquot 10mL volumes into 35 mm petri dishes and let stand at room temperature to solidfy. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept 10%</td>
			<td>To make 100mL: 10g Tegosept 100 mL Ethanol</td>
			<td>Store aliquots at -20 degrees C</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast paste</td>
			<td>~50 g Dry Active Yeast</td>
			<td>Gradually add ddH2O to beaker containing yeast while stirring until paste-like consistency reached. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2: Staining Materials</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>D3571 //// Stock at 5mg/mL</td>
		</tr>
		<tr>
			<td>rabbit anti-Vasa</td>
			<td>1:250</td>
			<td></td>
			<td>A gift from Ruth Lehmann</td>
		</tr>
		<tr>
			<td>mouse anti-Fasciclin III</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>7G10</td>
		</tr>
		<tr>
			<td>mouse anti-1B1</td>
			<td>1:4</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>1B1</td>
		</tr>
		<tr>
			<td>guniea pig anti-Traffic Jam</td>
			<td>1:2500</td>
			<td></td>
			<td>A gift from Dorthea Godt (Li et al, 2003)</td>
		</tr>
		<tr>
			<td>mouse anti-Prospero</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>Prospero MR1A</td>
		</tr>
		<tr>
			<td>rat anti-Elav</td>
			<td>1:30</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>Rat-elav 7EBA10 anti-elav</td>
		</tr>
		<tr>
			<td>mouse anti-Repo</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>8D12 anti-Repo</td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa546</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11010</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa488</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11029</td>
		</tr>
		<tr>
			<td>goat anti-guniea pig Alexa633</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A21105</td>
		</tr>
		<tr>
			<td>goat anti-rat Alexa488</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11006</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3: Materials and Equipment</td>
		</tr>
		<tr>
			<td>Fly Cages</td>
			<td>Hand-made; Genesee Scientific Corporation</td>
			<td>Not applicable; Bottles: 32-130; Pre-made cage: 59-101</td>
			<td>Made by cutting clear cast acrylic tubing (1 3/4 inch in diameter) into 4 inch tall segments with a compound miter saw at 400 rpm. Ultrafine stainless steel screening (was attached to one end of the tub with acrylic compund glue. An alternate method using an empty fly food bottle can be found in Drosophila Protocols ISBN 0-87969-584-4. Cages may also be purchased from the Genesee Scientific Corporation.</td>
		</tr>
		<tr>
			<td>Sonicator: Branson 250 Digital Sonifier</td>
			<td>Branson</td>
			<td>Model: Branson Digital Sonifier 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator Probe</td>
			<td>Branson</td>
			<td>Model #: 102C (CE)</td>
			<td>EDP: 101-135-066; S/N: OBU06064658</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Nalgene</td>
			<td>190-25-20</td>
			<td>0.2 micrometer cellulose, acetate membrane filter</td>
		</tr>
		<tr>
			<td>Imaging system: Spinning disc confocal microscope with multichromatic light source, digital CCD camera, and imaging software</td>
			<td>Microscope: Olympus Light source: Lumen Dynamics Camera: Q-Imaging Imaging Software: Intelligent Imaging Inc.</td>
			<td>Microscope: BX51 equipped with DSU spinning disc Light source: X-Cite 120Q Camera: RETIGA-SRV Imaging Software: Slidebook 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter unit</td>
			<td>Nalgene</td>
			<td>450-0020</td>
			<td>0.2 micrometer cellulose nitrate membrane filter</td>
		</tr>
	</tbody>
</table>

sonication-facilitated immunofluorescence staining of late-stage embryonic and larval drosophila tissues in situ

מחקרים שבוצעו בתסיסנית עוברים וזחלים מספקים תובנה חיונית לתהליכים התפתחותיים כגון מפרט גורל התא וorganogenesis. Immunostaining מאפשר ההדמיה של פיתוח רקמות ואיברים. עם זאת, לציפורן הגנה שיוצרת בסופו של עובר מונעת חלחול של נוגדנים לעוברי בשלב מאוחר וזחלים. בעוד לנתיחה לפני immunostaining משמשת באופן קבוע לנתח רקמות זחל דרוזופילה, זה מוכיח לא יעיל עבור חלק ניתוחים בגלל רקמות קטנות עשויות להיות קשות לאתר ולבודד. Sonication מספק חלופה לנתיחה בפרוטוקולי immunostaining דרוזופילה זחל. זה מאפשר עיבוד מהיר, בו זמני של מספר הגדול של עוברים בשלב מאוחר וזחלים ושומר במורפולוגיה באתר. לאחר הקיבוע בפורמלין, מדגם sonicated. לדוגמא נתונה אז immunostaining עם נמלה העיקרית אנטיגן ספציפיibodies ונוגדנים משני שכותרתו fluorescently לדמיין סוגי תאי היעד וחלבונים ספציפיים באמצעות מיקרוסקופ פלואורסצנטי. במהלך התהליך של sonication, מיקום נכון של בדיקה sonicating מעל המדגם, כמו גם את המשך ועוצמת sonication, הוא קריטי. שינויים קלים additonal לפרוטוקולי immunostaining סטנדרטיים עשויים להידרש לכתמים באיכות גבוהות. לנוגדנים עם אות נמוכה יחס רעש, פעמים דגירה ארוכות יותר הן בדרך כלל צורך בכך. כהוכחה של קונספט לפרוטוקול הקל sonication זה, אנו מראים immunostains של שלושה סוגי רקמות (אשכים, השחלות, ורקמות עצביות) בטווח של שלבי התפתחות.

כדי להדגים את היעילות של immunostaining מבוסס sonication בניתוח עוברי בשלב מאוחר וברקמות זחל באתר, עוברי wild-type וזחלים עובדו לimmunostaining של אשכים, השחלות, ורקמה עצבית. דגימות הם צילמו באמצעות מיקרוסקופיה confocal ותוצאות נציג מוצגות (איור 1 ואיור 2). תוצאות עולות כי הפרוטוקו...

Watch this Scientific Journal Video about Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ at JoVE.com

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

הקל Sonication Immunofluorescence הכתמה של שלב מאוחר עוברי וזחל<em&gt; דרוזופילה</em&gt; רקמות<em&gt; באתר</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

12.5K Views.  College of William & Mary. Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae. 

Video: Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection של מערכת העצבים המרכזית הזחל ב תסיסנית melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

תרבויות עצבית ראשונית של המוח של Late Stage תסיסנית גלמים

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

תסיסנית הזחל Dissection NMJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

תסיסנית הזחל NMJ אימונוהיסטוכימיה

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

הכנת עוברים אלקטרונים מיקרוסקופית של תסיסנית לב עוברי צינור

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Microdissection של רקמות דג הזברה העין עובריים

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissection ו מכתים של תסיסנית הזחל השחלות

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

ניתוח של עוברי וזחל myofibers שלד דג הזברה מהכנות ניתקו

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

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 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

מדידה של הזחל פעילות ב<em&gt; דרוזופילה</em&gt; צג פעילות

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...