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).

注意:程序在室温下，除非另有说明进行。温度控制的恒温箱用于维持温度为蝇饲养和跨越除非另有说明。 1.准备<ol><li>准备苍蝇。 <ol><li>前中期富集的卵母细胞的集合。 <ol><li>清除成年健康，年轻的股票或跨文化的苍蝇。年龄瓶在25℃两天。 注:一般两个健康的瓶子就足够了，但更多的可能需要一些交叉文化。 </li><li>两天后，收集来自瓶〜100〜300位女性（谁在这一点上是0〜2日龄）。女性并不需要是处女。酵母膏的轻拍添加到小瓶的侧面，并放置30名女性和每次10〜15雄性成yeasted小瓶中。年龄小瓶中在25℃两天。 </li></ol></li><li>中期富集的卵母细胞集合。 <ol><li>收集股票或跨文化性〜100〜300位女性上课。酵母膏的轻拍添加到小瓶的侧面，并放置30位女性的每个（与没有添加男性）插入yeasted小瓶中。年龄小瓶为三到五天在25℃。 </li></ol></li></ol></li><li>准备解决方案。 注意:溶液可无限期地在室温下保存，除非另有说明。 <ol><li>制备改性罗伯的缓冲液（5×）:500毫米的HEPES，500mM的蔗糖，275 mM乙酸钠，200mM的乙酸钾，50mM葡萄糖，6mM氯化镁，和5mM氯化钙。使用10 N 11:8氢氧化钠:氢氧化钾，使pH值至7.4。通过过滤消毒;不要高压。储存于-20℃。根据需要准备〜200毫升1X罗伯的每卵母细胞准备的解冻。 </li><li>固定解决方案。 <ol><li>选项1:准备甲醛/庚烷固定。准备固定缓冲液:1×磷酸盐缓冲盐水（PBS）加150毫蔗糖。要使用，请与687.5微升固定缓冲液和312.5微升16％的甲醛清新每个卵母细胞准备。 注意事项:当在通风橱中使用甲醛溶液戴手套。根据机构指引的废弃物处理。 </li><li>选项＃2:准备甲醛/二甲胂固定。准备修复混合:250mM蔗糖，100mM乙酸钾（pH7.5）中，25 mM乙酸钠（pH 7.0），和25mM EGTA（pH 8.0）中。使用，使新鲜用400μl修复混合，加入100μl二甲胂酸钾（1M，pH7.2）中，并每卵母细胞制备500μl的16％的甲醛。 注意:二甲胂酸钾含有砷。 </li></ol></li><li>制备PBS /的Triton X-100:1×PBS加1％或0.05％的Triton X-100。保存在4℃。 </li><li>制备PBS-吐温20牛血清白蛋白（BSA）（PTB）:1×PBS中，0.5％BSA（重量/体积）和0.1％吐温-20。可以被存储在4℃下一周。 </li><li>荧光原位杂交（FISH）解决方案。 <ol><li>制备20X氯化钠-柠檬酸钠（SSC）:3M氯化钠和0.3M的柠檬酸钠。 </li><li>制备2×SSC-吐温20（SSCT）:2×SSC加0.1％吐温-20。让每个卵母细胞准备新鲜，〜20毫升</li><li>制备酰胺的解决方案:2X SSC，0.1％吐温-20，加甲酰胺。使新鲜，将1ml 20％的甲酰胺加入0.5ml 40％的甲酰胺，和每卵母细胞制备2毫升50％甲酰胺。 注意事项:当在通风橱中使用甲酰胺的解决方案戴上手套。根据机构指引的废弃物处理。 </li><li>制备杂交溶液:2×SSC，50％甲酰胺，和10％硫酸葡聚糖（重量/体积）。保存在4℃。 </li></ol></li><li> FISH探针。 <ol><li>为了寡核苷酸（见序列表1）用HPLC纯化和所需的5&#39;荧光修改（ 例如 ，Cy3标记或Cy5的）。重悬在Tris-EDTA（TE），在50毫微克/微升。 注:在任何时候都曝光保护寡核苷酸。 </li></ol></li></ol></li></ol> 2.晚期果蝇卵母细胞的收集<ol><li>如<eM&gt;果蝇卵母细胞的膜完整会粘到塑料和玻璃，预涂层1个五毫升管和每卵母细胞制备1巴斯德吸管与PTB的内部。 </li><li>所有的麻醉约100至300 yeasted苍蝇与二氧化碳，并添加到含有约100ml 1X罗伯的缓冲搅拌机。脉冲三次（〜每1秒）。请罗布的卵母细胞&lt;20分钟，以避免激活。 注:另外，卵母细胞可从女性手工解剖。这种方法的优点是，它需要较少的女性。但是，必须小心，以暴露于二氧化碳限制为仅几分钟，以避免与缺氧7相关联的伪影。 </li><li>通过大目（〜1500微米）到容量250ml的烧杯过滤器除去大的身体部位。如果许多完好的腹部仍然在使用额外的罗伯的缓冲滤网，再研磨材料，并再次。让定居〜2分钟，然后吸掉上层，删除尽可能多的大的身体部位尽可能的。 </li><li>网络连接滤波器通过小目（〜300微米）到250ml烧杯中。冲洗剩余的卵母细胞出使用额外的罗伯的和涂层巴斯德吸管第一烧杯中。让落户〜3分钟;卵母细胞会沉淀下来。吸掉所有，但〜10毫升</li><li>倒尽可能多的10毫升与适合涂5毫升管。让我们平静下来，去除液体，并用剩下的重复。冲洗剩余的卵母细胞出使用额外的罗伯的和涂层巴斯德吸管烧杯中。让定居在5毫升试管〜3-5分钟。 </li></ol> 3.药物治疗（可选） <ol><li>大衣2个5毫升管与PTB每个卵母细胞的准备。合适的溶剂（对照）或药物添加到1ml罗布的每一个用于每个卵母细胞制备（ 表2）。 </li><li>卵母细胞分裂成第二涂层5毫升管。让我们平静下来，除去液体，加入1 ml罗伯的加溶剂到一个试管和1ml罗布的加药物进入第二管。章动的时间为药物治疗（ 表2）适当的量。大号等安顿。 </li></ol> 4.固定<ol><li>吸掉所有的液体，并立即加入1ml修复。 </li><li>选项1:甲醛/庚烷固定（固定缓冲液加5％甲醛）。 <ol><li>修正了一个章动器2.5分钟。加入1 ml庚烷和涡旋1分钟。让沉降〜1分钟。 </li><li>删除所有的液体，然后再加入1 ml 1X PBS。旋涡30秒。让沉降〜1分钟。 </li><li>删除所有的液体，然后填写管1X PBS。 注:卵母细胞可以立即使用或保存在章动器在室温下几个小时。 </li></ol></li><li>选项2:甲醛/二甲胂固定（固定混合加8％的甲醛和100毫米二甲砷酸盐）。 <ol><li>修正了一个章动器6分钟。让定居2分钟。去除液体，然后填写管1X PBS。 注:卵母细胞可以立即使用或保存在章动器在室温下几个小时。 </li></ol></li></ol> 5.去除膜（"滚动"） <ol><li>使用涂覆巴斯德吸管，添加〜500〜1000的卵母细胞于载玻片的磨砂（喷砂）的一部分。删除所有身体部位和外来物质的使用镊子。不要让卵母细胞干出来的;加1×PBS中是必要的。 </li><li>放置在卵母细胞，并轻轻"滚"卵母细胞中，直到所有的膜的顶部上的盖玻片除去（拖动盖玻片边缘穿过卵母细胞的效果最好。）定期检查显微镜下进展，根据需要加入更多的1倍PBS中。小心为过大的压力会破坏卵母细胞。 注:对于只免疫，继续步骤6鱼（有或无免疫），请继续第7步。 </li></ol> 6. 果蝇卵母细胞的抗体染色<ol><li>提取和阻塞<ol><li>冲洗轧制的卵母细胞成含有约15毫升的PBS / 1％的Triton X-100的15毫升锥形管中。章动的卵母细胞在该溶液中不低于1.5小时，不超过2小时。此步骤允许抗体渗透。 </li><li>让卵母细胞定居〜2分钟。与尽可能多的膜尽量沿去除液体，再加入PBS / 0.05％的Triton X-100。 注:膜将比推出的卵母细胞定居慢。 </li><li>让卵母细胞定居〜2分钟，然后取出所有，但约1毫升的液体。转移卵母细胞毕业1.5mL管中，并去除残留液体。加入1ml的PTB用于阻断和章动1小时。 </li></ol></li><li>抗体染色<ol><li>抗胚胎次级抗体的预吸收。 注:此步骤从果蝇蛋白的非特异性抗体相互作用消除了背景染色。 <ol><li>收集和固定每个典型的程序8 果蝇胚胎（〜25微升）。在-20℃储存在甲醇。 </li><li>从胚胎中除去甲醇，加入800微升甲醇加200微升1×PBS中，并且章动15分钟。除去500μl的上清液，用500μl1×PBS中替换。然后章动15分钟。重复两次（总共洗涤约1小时的），然后在PTB完成。 注:胚胎可以立即使用或保存在章动器在室温下几个小时。 </li><li>除去液体，用PTB填充到每卵母细胞制备200微升，并在适当的稀释液添加荧光标记的二抗（牢记最终体积为300微升;参见步骤7.4）在4℃章动过夜（优选）或在室温下进行3至4小时。 注意:预吸收抗体将在步骤6.2.4使用。 注意:保持样品在黑暗中尽可能一次荧光抗体已被添加。 </li></ol></li><li>从卵母细胞除去液体，具有PTB补到300微升，并在适当的稀释液添加的主要抗体。章动于4℃过夜（优选），或在室温下搅拌3至4小时。 </li><li>洗涤卵母细胞四次用1ml PTB每次15分钟。 </li><li>去掉从卵母细胞的液体，从胚胎（预吸收，二次抗体）加入200μl上清液。然后用PTB填充到300微升。章动在室温下搅拌3〜4小时（优选），或在4℃下过夜。 </li><li> 15分钟洗一次卵母细胞用1毫升PTB，去除液体。然后添加0.5微升的Hoechst 33342和500μl的PTB和章动7分钟。 </li><li>洗的卵母细胞两次，每次15分钟，用1ml PTB。 注:卵母细胞可以立即安装在滑动或可在4℃储存在PTB直至准备成像。 </li></ol></li></ol> 7. FISH（从上面的步骤5继续） <ol><li>冲洗轧制的卵母细胞成含有〜15毫升2×SSCT 15毫升锥形管中。让卵母细胞定居〜2分钟。删除所有，但约0.5毫升的液体随着许多膜越好。 注:膜将比推出的卵母细胞定居慢。 </li><li>卵母细胞转移到0.5毫升管和去除残留液体。先后添加和删除500微升20％，40％，和50％甲酰胺溶液，章动在每个溶液10分钟。 注:卵母细胞将落户较慢甲酰胺的比例较高。 </li><li>除去液体，然后在37℃下加入500μl50％甲酰胺溶液，并章动进行1至5小时。 注意:长时间孵育带来更好的探头渗透。 </li><li>去除液体，留下不超过〜100微升的卵母细胞，并加入36微升的杂交溶液加2微升探针（50毫微克/微升）和2微升的水或2微升第二探针（ 表1）。 </li><li>孵育91℃3分钟（FISH只），或80℃20分钟（FISH加免疫荧光），接着温育在37℃水浴中过夜。 注:可在热循环中执行这些步骤。 </li><li>不要去除液体。加入500微升50％甲酰胺溶液和章动，在37℃1小时。 </li><li>让我们平静下来，去除液体，加入500μl50％甲酰胺溶液中，在37℃下1小时ð章动。 </li><li>让沉降，除去液体，在室温下加入500μl20％甲酰胺溶液，并章动10分钟。 </li><li>在500微升2倍SSCT执行三个快速冲洗（让落户，取出然后加液，颠倒几次，重复。）让定居，去除液体，然后加入500μlPTB和章动4小时。 </li></ol> 8.抗体染色后鱼<ol><li>从卵母细胞去掉液体，具有PTB补至300微升，并加入缀合至FITC 10微升抗α微管蛋白抗体。可替代地，其它抗体，可以使用按照上述的协议。章动在室温下过夜。 </li><li>对于用500μlPTB 15分钟洗的卵母细胞后，除去液体，再加入0.5微升的Hoechst 33342和500μl的PTB和章动7分钟。 </li><li>与500微升PTB每次15分钟洗涤两次卵母细胞。 注:卵母细胞可以立即安装在滑动或可在4℃储存在PTB直至准备成像。保持卵母细胞在黑暗中尽可能一次荧光抗体已被添加。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1in"> 重复名称 </td><td fo:width="1in"> 染色体 </td><td> 寡核苷酸序列* </td></tr><tr><td> 359 </td><td> X </td><td> GGGATCGTTAGCACTGGTAATTAGCTGC </td></tr><tr><td> AACAC </td><td> 2 </td><td> AACACAACACAACACAACACAACACAACACAACACAACAC </td></tr><tr><td>十二</td><td> 3 </td><td> CCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT </td></tr><tr><td> 1.686 </td><td> 2 + 3 </td><td> AATAACATAGAATAACATAGAATAACATAG </td></tr><tr><td> AATAT </td><td> 4（+ Y） </td><td> AATATAATATAATATAATATAATATAATAT </td></tr><tr><td colspan="3"> *从埃里克·乔伊斯，个人通信，沙利文等其他序列359序列。8 </td&gt; </tr></tbody></table> 表1:FISH 探针果蝇 着丝粒重复。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 药物 </td><td> 溶剂 </td><td> 股权集中度 </td><td> 最终浓度 </td><td> 治疗时间 </td><td> 影响 </td></tr><tr><td>秋水仙碱</td><td>乙醇</td><td> 125毫米</td><td> 150μM </td><td> 10分钟或30分钟</td><td>破坏非着丝粒（10分钟）9或所有（30分钟）的微管</td></tr><tr><td>紫杉醇</td><td> DMSO </td><td> 10毫</td><td> 10μM </td><td> 10分钟</td><td>稳定话筒rotubules </td></tr><tr><td> Binucleine 2 </td><td> DMSO </td><td> 25毫米</td><td> 25μM </td><td> 20分钟</td><td>抑制极光酶b激酶10 </td></tr></tbody></table> 表2:药物治疗。

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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.

分期果蝇卵母细胞 虽然细长karyosome常见于前中期的卵母细胞，用karyosome形状从中期的卵母细胞前中期区分可能会有问题。期间前中期，所述karyosome开始表现为圆形，伸长，然后作为卵母细胞接近中期阻滞缩回到圆形。这意味着许多前中期的卵母细胞不具有细长karyosome。此外，如果被检查体或药物处理的卵母细胞，karyosome形状可能受到影响，使用这种方法?...

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.

将导致较减数分裂的三个阶段（ 图1）的后期果蝇卵母细胞的集合中，我们在这里描述的方法。在前期的卵母细胞通过核膜的存在，这是由在karyosome（ 图1A）周围区域缺乏微管蛋白信号的可见区别。前中期是核膜破裂期间，主轴组装后的时期。期间前中期，所述karyosome呈现独特的形状，成为与从主karyosome质量（ 图1B）的第</s...

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.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

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 次观看.  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.

视频: 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.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

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

科研

生物学

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.

为介观荧光体层摄影术在体内成像的发展果蝇</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.

发展和成人的制备果蝇大脑和视网膜实时成像

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.

技术成像钙 2 +信号在人类精子

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.

直立成像果蝇胚胎

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.

在完整的果蝇幼虫体内的亚细胞分辨率成像

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.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

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.

影像生活细胞的形状变化果蝇胚胎

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.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

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.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

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