We thank Christian Lehner for providing the CENP-C antibody and Eric Joyce for recommendations on FISH. Work in the McKim lab was funded by a grant from NIH (GM101955).

<ol>
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D., 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=Hypoxia+transiently+sequesters+mps1+and+polo+to+collagenase-sensitive+filaments+in+Drosophila+prometaphase+oocytes.">Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.</a> PLoS One. 4 (10), e7544 (2009).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Radford, S. J., Hoang, T. L., G&#322;uszek, A. A., Ohkura, H., McKim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+and+End-On+Kinetochore+Attachments+Are+Coordinated+to+Achieve+Bi-orientation+in+Drosophila+Oocytes.">Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.</a> PLoS Genet. 11 (10), e1005605 (2015).</li><li>Smurnyy, Y., Toms, A. V., Hickson, G. R., Eck, M. J., Eggert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binucleine+2,+an+isoform-specific+inhibitor+of+Drosophila+Aurora+B+kinase,+provides+insights+into+the+mechanism+of+cytokinesis.">Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis.</a> ACS Chem Biol. 5 (11), 1015-1020 (2010).</li><li>Mahowald, A. P., Goralski, T. J., Caulton, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activation+of+Drosophila+eggs.">In vitro activation of Drosophila eggs.</a> Dev Biol. 98 (2), 437-445 (1983).</li><li>Page, A. W., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+meiotic+divisions+in+Drosophila+oocytes.">Activation of the meiotic divisions in Drosophila oocytes.</a> Dev Biol. 183 (2), 195-207 (1997).</li><li>Tavosanis, G., Llamazares, S., Goulielmos, G., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+gamma-tubulin+in+the+acentriolar+female+meiotic+spindle+of+Drosophila.">Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.</a> EMBO J. 16 (8), 1809-1819 (1997).</li><li>Endow, S. A., Komma, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+dynamics+during+meiosis+in+Drosophila+oocytes.">Spindle dynamics during meiosis in Drosophila oocytes.</a> J Cell Biol. 137 (6), 1321-1336 (1997).</li><li>Matthies, H. J., Clarkson, M., Saint, R. B., Namba, R., Hawley, R. S., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , 67-85 (2000).</li><li>Colombi&#233;, N., 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=Dual+roles+of+Incenp+crucial+to+the+assembly+of+the+acentrosomal+metaphase+spindle+in+female+meiosis.">Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.</a> Development. 135 (19), 3239-3246 (2008).</li></ol>

The authors have nothing to disclose.

Staging תסיסנית ביציות אם כי karyosome מוארך נראה בדרך כלל ב ביציות prometaphase, באמצעות karyosome צורה להבחין prometaphase מ ביציות metaphase יכול להיות בעייתי. במהלך prometaphase, את karyosome מתחיל כמו צורה עגולה, מתארך, ולאחר מכן חזר בו כדי צורה עגולה כמו הביצית מתקרבת מעצר metaphase. משמעות הדבר היא כי רבים ביציות prometaphase אין לי karyosome מוארך. בנוסף, אם ביציות מוטציה או מטופלים בתרופה נבחנות, karyosome צורה עלולה להיפגע, לא ניתן בשיטה זו כדי לביים ביציות. מכיוון סמנים אחרים להבחין בשלבים אלה אינם זמינים כרגע, אנו משתמשים בשיטה המתוארת לעיל כדי לתפעל את המהירות-ההטלה של נקבות תסיסנית להעשיר עבור ביציות בין אם prometaphase או metaphase. הרציונל הוא שאם ביציות להשקיע סכום מסוים של זמן prometaphase, וכן ללא הגבלת זמן עיבודested ב metaphase, שנמשך עד הטלה, אז יותר-הטלה מהירה יהיה להטות כלפי אחוז גבוה יותר של ביציות prometaphase, תוך-הטלה איטית תעשיר עבור metaphase. כמות המאמץ הנדרשת כדי לאסוף נקבות בתולה או לאסוף נקבות על מסגרת זמן קצרה יותר מ -2 ימים, כפי שתוארה על ידי Gilliland et al. 6, הוא לא מתוגמל על ידי גידול ניכר ביציות prometaphase. זה גם בלתי אפשרי להשתמש נספחים הגבו לביים ביציות מוכנות עם פרוטוקול זה כנספחים הגבו יוסרו יחד עם ממברנות. למרות זאת הוא לא ניתן לביים ביציות בודדות עם 100 ודאויות% בשיטה זו, הנתונים מן המועשר prometaphase או אוספים מועשרים metaphase ניתן לקחת יחד כדי להסיק מסקנות לגבי השלבים השונים הללו 9. ביציות תסיסנית אינן מופרות עד שהם עוברים דרך oviduct; ולכן, בגלל הביציות שנאספו עבור הפרוטוקולי דואר המתואר כאן לבחון ביציות שנלקחו לפני הטלת ביציות אלו לא הופרו. המעבר דרך oviduct גם גורם הביצית כדי להפעיל ולחדש התקדמות מחזור התא. שיטות לבחון בשלבי מחזור התא לאחר metaphase אני קיים 11,12, אך הן מחוץ להיקף של פרוטוקול זה. שיקולי קיבוע שיטה אנו מתארים שתי שיטות נפרדות עבור הקיבעון של ביציות תסיסנית בשלב מאוחר. שיטת קיבוע פורמלדהיד / cacodylate תוארה לראשונה על ידי Theurkauf ואת האולי 3 ואת שיטת קיבוע פורמלדהיד / heptane תוארה לראשונה על ידי Zou et al. 4. בנוסף, שיטה שלישית (קיבעון מתנול) משמשת ב -13 מסוימים, אבל לא המתוארת כאן. הסרת ממברנות הביצית היא קצת יותר קשה לאחר קיבוע פורמלדהיד / heptane לעומת פורמלדהיד / cacodylate. הסיבה לכך היא &quot;מתגלגל&quot; תלויההים על חיכוך בין coverslip ואת הביצית, קיבעון פורמלדהיד / heptane עושה הביציות חלקלקות יותר. למרות הקושי המוסף הזה, הבחירה הראשונה שלנו של שיטת קיבוע כאשר בודקים נוגדנים חדשים הוא פורמלדהיד / heptane מכיוון שהיא מתאימה ביותר עבור רוב הנוגדנים שניסינו. מצד השני, אנו מעדיפים קיבוע פורמלדהיד / cacodylate לדגים, בעיקר ההקלות של הסרת קרום, למרות קיבעון פורמלדהיד / heptane גם הצליח. שתי שיטות אלה לשמר מורפולוגיה ביצית טובה יותר קיבעון מתנול, ולכן אנו ממליצים קיבעון מתנול רק במקרים של נוגדנים הם עקשנים לשיטות קיבעון משחררי פורמלדהיד. הפרוטוקולים המתוארים כאן להתמקד הדמיה קבועה, על מנת להדגים שיטות יעילות עבור עיבוד ביציות תסיסנית בוגרת מקובל חדירת נוגדן, כלומר באמצעות הסרת ממברנות הביצית. שיטות חלופיות עבור קרום רםval (באמצעות מלקחיים 14 או sonication 13) אפשריים; עם זאת, אנו מוצאים &quot;מתגלגלים&quot; להיות השיטה היעילה והאמינה ביותר. טכניקות הדמיה לחיות גם תוארו במקומות אחרים 14-16 ו הן מחוץ להיקף של פרוטוקול זה. המלצות FISH דגי ביציות תסיסנית תוארו לראשונה על ידי Dernburg et al. 5. פרוטוקול זה תוכנן כדי לספק תנאים אופטימליים הכלאת בדיקה כדי הכרומוזומים הביצית המרוכזים, אבל הפרוטוקול המקורי לא היה אופטימלי עבור immunofluorescence. השיטה המתוארת לעיל כולל עיבודים עשינו כדי לשפר מכתים נוגדן של α-טובולין, וזה בעיקר מה אנו משתמשים בעת ביצוע FISH. עבור נוגדנים אחרים, הורדת הטמפרטורה של צעד denaturation (שלב 7.5) מ 80 ° C עשוי להיות נחוץ. הבדיקות המפורטות כאן הן עבוררצפים חוזרים של heterochromatin ליד צנטרומר. בגלל הרצפים אלה הם מאוד החוזרות על עצמן, החלליות לחשל למקומות מרובים ואת האותות הקולקטיביים די חזקים. יש לנו הצלחה קטנה עם בדיקות עבור רצפים ב euchromatin, ואולי בגלל המבנה הייחודי של הכרומוזומים בעוד כלול karyosome המתומצתת. הַדמָיָה ביציות תסיסנית בוגרות, כמו הביציות של רוב האורגניזמים, הם גדולים, תאים בודדים. כלומר יש נפח גדול של הציטופלסמה המכיל גרעין יחיד. למרבה המזל, גרעין זה, הידוע גם בתור karyosome תסיסנית, ניתן לזהות בקלות כפי שהוא בדרך כלל ממוקם בדיוק מתחת הנספחים הגבו הקרובים לקליפה. הנספחים הגבו יוסרו יחד עם ממברנות הביצית, אך karyosome יכול עדיין להיות ממוקם על ידי מציאת divot הקטן בקליפת הביצית שבו הנספחים oNCE היה. התמונות הטובות ביותר מושגות מתוך ביציות כי כבר רכוב עם השקע הזה מול coverslip מאז זה מציב את karyosome הקרוב המטרה. למרות שזה עשוי להיות אפשרי לארגן הביציות בכיוון הזה לפני ההרכבה, אנחנו מעדיפים לעלות ביציות רבות באופן אקראי ולאחר מכן לחפש את השקופית עבור אלה בכיוון המועדף. יישומים עתידיים בעתיד זה יהיה חשוב לזהות סמנים כי באופן מהימן להבחין prometaphase אני metaphase לי צירים. יכולת בצורה מהימנה יותר לביים הביציות תפחית את ההסתמכות על חלופות עבודה אינטנסיבית יותר, כגון הדמית מספר גדול של ביציות מועשר במה או הדמיה לחיות, שיש לה גם בעיות הבימוי שלו. שיטה זו היא השיטה הבסיסית אשר ניתן להשתמש בהם כדי לנצל את ההתקדמות בגנטיקה תסיסנית ככלים חדשים מתפרסמים לשנות או לדפוק מוצרי גן. זה כולל שיטותלמקד חלבון פעילות ישירות עם תרופות חדשות או אסטרטגיות השפלה ממוקדות. בנוסף, שיטות אלה אינם מוגבלים הדמיה הצירים meiotic. כל מבנה בתוך הביצית, כגון שלד תא יקטין, יכול להיות מנותח באמצעות שיטות אלה.

The study of meiosis is sometimes described as the "genetics of genetics". This is because the fundamental properties of chromosome inheritance and independent assortment are carried out through the segregation of chromosomes during gamete production. An important demonstration of the chromosome theory of inheritance came in 1916 from the work of Calvin Bridges in Drosophila melanogaster1. This and other classical genetics studies in Drosophila contributed greatly to our understanding of genetics. Cytological examination of meiotic chromosomes in Drosophila oocytes, however, has been challenging. This is primarily because immunofluorescence of late-stage Drosophila oocytes, when the spindle assembles and chromosomes are oriented for segregation, is hampered by the presence of membranes that render the oocyte impenetrable to antibodies.
Despite this challenge, Drosophila oocytes remain an attractive model for the study of chromosome behavior and spindle assembly. This is because of the powerful genetic tools available in Drosophila, but also because the oocytes arrest at metaphase I, when the chromosomes are oriented and the spindle is fully formed. This facilitates the collection and examination of large numbers of oocytes at this important stage of cell division. In addition, a simple model organism that is amenable to genetic manipulation for the study of oocyte chromosome segregation can provide an important contribution to our understanding of human reproductive health. Errors in chromosome number are the leading genetic cause of miscarriage and birth defects in humans2. A majority of these errors can be traced to the oocyte and are correlated with increasing maternal age. The average age of mothers in the U.S. has been increasing, making this a major public health concern.
We describe here methods for the cytological examination of Drosophila oocytes, including a demonstration of how to remove the oocyte membranes. These methods are modifications of protocols first described by Theurkauf and Hawley3, Zou et al.4, and Dernburg et al.5. We also include methods for the enrichment of different stages of oocytes, based on a protocol first described by Gilliland et al.6. Finally, we add instructions for the drug treatment of Drosophila oocytes. Together, these methods allow the cytological investigation of oocyte chromosome segregation and spindle assembly in Drosophila.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>HAZARDOUS; once opened, discard after one month</td>
		</tr>
		<tr>
			<td>250 mL beakers</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>active dry yeast</td>
			<td>Various</td>
			<td></td>
			<td>mix with water to make a paste the consistency of peanut butter</td>
		</tr>
		<tr>
			<td>anti-&#945;-tubulin antibody conjugated to FITC</td>
			<td>Sigma</td>
			<td>F2168</td>
			<td>clone DM1A</td>
		</tr>
		<tr>
			<td>Binucleine 2</td>
			<td>Sigma</td>
			<td>B1186</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>blender</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>colchicine</td>
			<td>Sigma</td>
			<td>C-9754</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>coverslips</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td>No. 1 1/2</td>
		</tr>
		<tr>
			<td>dextran sulfate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Ted Pella, Inc.</td>
			<td>5622</td>
			<td>Dumont tweezers high precision grade style 5</td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>Sigma</td>
			<td>47670-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>graduated 1.5 mL tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>EM-5330</td>
			<td>available from several venders</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>large mesh (~1500 &#181;m)</td>
			<td>VWR</td>
			<td>AA43657-NK</td>
			<td>variety of formats and other suppliers, 12 or 14 mesh</td>
		</tr>
		<tr>
			<td>small mesh (~300 &#181;m)</td>
			<td>Spectrum labs</td>
			<td>146 424</td>
			<td>variety of formats, eg 146 422 or 146 486</td>
		</tr>
		<tr>
			<td>nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>potassium acetate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cacodylic acid</td>
			<td>Sigma</td>
			<td>C0125</td>
			<td>HAZARDOUS; alternatively, sodium cacodylate may be substituted</td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium citrate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>taxol (paclitaxel)</td>
			<td>Sigma</td>
			<td>T1912</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>PI-28314</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher</td>
			<td>PI-28320</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

השיטות שתיארנו כאן תוביל גביית ביציות תסיסנית בשלב מאוחר המייצגים שלושה שלבי המיוזה (איור 1). ביציות ב prophase מאופיינים בנוכחות מעטפת הגרעין, אשר גלוי על ידי חוסר האות טובולין באזור המקיף את karyosome (איור 1 א). Prometaphase היא התקופה לאחר התמוטטות מעטפת גרעין שבמהלכו מרכיב הכישור. במהלך prometaphase, את karyosome לובשת צורה ייחודית, נעשה ארוך מדי לעתים קרובות עם 4 הכרומוזומים ה מלבד המסה karyosome הראשי (איור 1B). Prometaphase מסכם עם metaphase, שבו ביציות תסיסנית לעצור באופן טבעי עד הביוץ. ב ביציות metaphase, את karyosome חזר בו לתוך צורה עגולה (איור 1 ג). אנו מתארים כאן מ &#39;ethods לתפעל את המהירות והטלת ביצים של נקבות תסיסנית להעשיר עבור ביציות בין אם prometaphase או metaphase. ביציות מאוספים prometaphase מועשרים לעתים קרובות להציג את karyosome מאורך (איור 2 א) תוך אוספי metaphase מועשר או אוספים מן הנקבות תסיסנית בתולה, אשר כמעט לחלוטין לחסל-הטלה, בעיקר להראות את karyosome העגול (איור 2 ב). מעניין, אוספים prometaphase מועשרים גם בדרך כלל להראות צירים הרבה יותר חזק, דבר המצביעים על כך הוא שינוי karyosome ואת הציר בין prometaphase ו metaphase תסיסנית ביציות 9. איור 3 מציג תמונות מייצגות משני הפרוטוקולים המתוארים כאן. נוגדנים נגד טובולין α (כדי להראות את הציר), CENP-C (להראות צנטרומר), ו INCENP (כדי להראות את הציר המרכזי) שמשו על ביציות קבועות עם FOrmaldehyde / heptane (איור 3 א). בדיקות עבור רצפים centromeric חוזרות על 2 כרומוזום nd (AACAC) ו -3 rd כרומוזום (dodeca) שימשו על ביציות קבוע עם פורמלדהיד / cacodylate ושיתוף מוכתם נוגדן α-טובולין לפי הפרוטוקול FISH (איור 3B). חללית AACAC מופיעה מספר מוקדים התקבצו בעוד חללית dodeca מופיעה כמוקד יחיד לכל כרומוזום. ביציות שטופלו 150 קולכיצין מיקרומטר במשך 10 דקות תוקנו עם פורמלדהיד / heptane (איור 3 ג). טיפול זה הוא מחסל את קינטוכור-microtubules שאינו ביותר, שתוצאתה כל microtubules פניית DNA ב מוקדי שכותרתו centromere. ביציות שטופלו 10 paclitaxel מיקרומטר במשך 10 microtubules מוגזם דקות המופע בציטופלסמה, אך השפעה מועטה על microtubules ציר (איור 3D). ביציות שטופלו 25 מיקרומטר Binucleine 2 במשך 20 דקות כדי לעכב הצג kinase B אורורה לאובדן מוחלט שלmicrotubules ציר (איור 3E). <img alt="איור 1" src="/files/ftp_upload/54666/54666fig1.jpg" /> איור 1: שלושה שלבים של ביציות תסיסנית בוגרת:. Prophase, prometaphase, ו Metaphase תמונות Confocal של ביציות לתסיסנית prophase (א), prometaphase (B), ואת metaphase (C) של ה- DNA I. המיוזה מוצג בכחול טובולין הוא מוצג בירוק בתמונות ממוזגות. ריבועים (ב) ו- (ג) להראות DNA בלבן. ברי סולם = 10 מיקרומטר. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54666/54666fig1large.jpg" target="_blank">לחצו כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/54666/54666fig2.jpg" /> איור 2:Prometaphase- לעומת קולקציות metaphase מועשר של ביציות תסיסנית. תמונות Confocal של ביציות תסיסנית מ- (A) prometaphase מועשר או מועשר metaphase (B) אוספים. DNA מוצג בכחול טובולין מוצג בירוק. ברי סולם = 10 מיקרומטר. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54666/54666fig2large.jpg" target="_blank">לחצו כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/54666/54666fig3.jpg" /> איור 3: מכתים נוגדן, דגים, טיפולים תרופתיים של ביציות תסיסנית תמונות Confocal של ביציות תסיסנית עם DNA בכחול טובולין בירוק.. ברי סולם = 10 מיקרומטר. (א) Immunofluorescence ב פורמליןקיבעון דואר / heptane עם INCENP (ציר מרכזי) באדום CENP-C (צנטרומר) בלבן. (ב) דגים קיבעון פורמלדהיד / cacodylate עם AACAC באדום ו dodeca המוצגים לבן. (ג) הטיפול עם 150 קולכיצין מיקרומטר במשך 10 דקות עם CENP-C באדום. טיפול עם 10 מיקרומטר paclitaxel במשך 10 דקות (D) או 25 מיקרומטר Binucleine 2 במשך 20 דקות (E). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54666/54666fig3large.jpg" target="_blank">נא ללחוץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

טכניקות הדמיה prometaphase ו Metaphase של המיוזה ואני קבוע<em&gt; תסיסנית</em&gt; ביציות

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

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Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Unit 2

Meiosis

SCIENTISTS IN ACTION

9.0K Views.  Rutgers University. We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Video: Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Microinjection טכניקות לימוד מיטוזה ב תסיסנית melanogaster סינסיציאלי עובריים

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopic Fluorescence טומוגרפיה עבור In-vivo הדמיה של פיתוח תסיסנית</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

הכנת פיתוח למבוגרים תסיסנית מוח רשתיות עבור הדמיה חיה

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

טכניקות הדמיה Ca 2 + איתות זרע אנושי

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

הדמיה של זקופה תסיסנית עוברים

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

בתחום ההדמיה vivo של הזחלים תסיסנית אינטקט ברזולוציה משנה הסלולר

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

צורה הדמיה תא שינוי חיים תסיסנית עוברים

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

חי הדמיה של חלבוני ה-GFP שהכותרת ב-<em&gt; דרוזופילה</em&gt; ביציות

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

חי הדמיה של חיסול תאים אפופטוטיים במהלך<em&gt; דרוזופילה</em&gt; עובר

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...