We are grateful to Chau-Ti Ting (Fly Core in Taiwan) for providing the monoclonal 4D9 antibody, Technology Commons (TechComm) of the College of Life Science NTU for confocal microscopy, Hsiao-Ling Lu for proofreading the manuscript, and Chen-yo Chung for helping filming. CC particularly thanks Charles E. Cook for providing strategic suggestions and for critical editing of the manuscript. This work was supported by the Ministry of Science and Technology (101-2313-B-002-059-MY3 and 104-2313-B-002-022-MY3 for GWL and CC), and the National Taiwan University (NTU-CESRP 101R4602D3 for CC; 103R4000 for GWL).

蚜虫1.文化注:孤雌胎生豌豆蚜A的实验室株豌豆最初收集在中央台和在长日照的光周期已经饲养的寄主植物（花园豌豆豌豆或蚕豆蚕豆 ）超过300代（一代:〜10天）。 <ol><li>种子发芽<ol><li>浸泡寄主植物的种子在自来水中3-5天在RT。加注新水，每天一次。 注:另外，把寄主植物的种子直接进入潮湿的土壤中可诱发发芽为好。 </li><li>增长10发芽的种子在小火锅（直径9厘米×7厘米高），与土壤中的生长室下光照16小时光照/ 8小时黑暗中，在20℃。 </li><li>生长开始后10天左右，蚜虫转移到植物的高度为8厘米以上。 </li></ol></li><li>蚜虫转移<o升&gt; <li>保持植物的每锅内的1L玻璃烧杯中，传输8成年蚜虫到使用画笔植物，然后密封与透气式盖烧杯如纱布目以防止蚜虫逸出。 </li><li>孵育在生长室蚜虫下光照16小时光照/ 8小时黑暗中，在20℃。每天每个水植物盆栽植物的一次。 </li><li>为了获得下一代蚜虫，重申步骤1.1.3-1.2.2主蚜虫转让后十天。 </li></ol></li></ol> 2.解剖和卵巢的固定<ol><li>新鲜在1×磷酸盐缓冲盐水（PBS）作为固定缓冲器制备4％低聚甲醛（PFA）。 </li><li>填充斑点板与PFA（约500微升）的一个孔中，将板在立体显微镜在低放大倍数下，和在PFA内淹没成年蚜虫解剖。 </li><li>握住头部和腹部有一组镊子，切开背解剖卵巢角质层腹部，并从腹腔拖动卵巢远。 </li><li>固定在1.5ml试管含有1ml的PFA在RT 20分钟3双卵巢。 </li><li>倒出固定缓冲用移液管（玻璃或一次性的），然后洗涤卵巢用0.2％的TritonX-100在1×PBS（PBST）中3次，每次10分钟。温和摇动上的混合器/旋转器（旋转角度:60°（F60）/转速:8 RPM）被推荐用于固定和洗涤。 </li></ol> 3.治疗与蛋白酶K（PK），以提高胚胎组织的通透性注:PK治疗是从胚芽带扩展名以后（发展阶段11）适用于胚胎。对于年轻的胚胎，这一步是可选的。 <ol><li>串联稀PK的原液（10毫克/毫升）用1×PBS至工作浓度1μg/ ml的。 </li><li>孵育卵巢与PK（约500微升）的1μg/ ml的10分钟，轻微摇动。 </li><li>倒出PK soluti上，然后用700微升甘氨酸（2毫克/毫升）3次，每次5分钟洗涤卵巢。 </li><li>每10分钟洗涤卵巢用0.2％PBST清洗两次。 </li><li>再次固定卵巢与固定缓冲液15分钟，室温，轻微摇动。 </li><li>弃上清，每10分钟洗涤卵巢用0.2％PBST清洗两次。 </li></ol> 4.甲醇孵化为了抑制内源性过氧化物酶的活性注:甲醇孵育没有施加到胚胎经受鬼笔环肽染色或抗体表位是甲醇敏感。 <ol><li>串联脱水卵巢与甲醇的0.2％PBST清洗不同的百分比（体积/体积:1:3,1:1,3:1）通过将卵巢在甲醇溶液各浓度进行10分钟适度搅拌。 </li><li>脱水卵巢用100％甲醇进行1小时，在室温，轻微摇动。 注:染色的满意的结果仍然可以从在存储在100％甲醇中那蚜组织得到-20＆＃176;下一个月。 </li><li>串联再水化卵巢与甲醇的0.2％PBST清洗不同的百分比（体积/体积:3:1，1:1,1:3）通过将卵巢在甲醇溶液各浓度进行10分钟适度搅拌。 </li></ol> 5.抗体染色<ol><li>稀释从该DIG基于缓冲器集的10倍封闭溶液（DIG-B）的1倍。 注:1×辛乙封闭溶液是更有效地减少染色背景比标准封闭试剂5％组成（体积/体积）正常山羊血清（NGS）和0.5％（体积/体积）牛血清白蛋白（BSA ）在0.2％PBST清洗。 </li><li>孵育1倍辛乙阻断为2.5至4小时，在室温或O / N溶液在4℃下，轻微摇动卵巢。 注:对于3对在1.5ml管卵巢，200微升是最小体积为样品阻挡和抗体染色。 </li><li>滗上清液和替换与含有适当diluti初级抗体新鲜的1X辛乙封闭溶液对比率。染色在RT或O / N卵巢4小时，在4℃，轻微摇动。 注:对于这里描述的实验中，使用初级抗体的以下最佳稀释度:（1）ApVas1抗体:1:500的显色染色，1:50免疫荧光染色; （2）抗α微管蛋白抗体:1:500; （3）4D9单克隆抗体:1:25。 </li><li>用0.2％PBST清洗，每次15分钟，洗涤卵巢4次。 </li><li>孵育卵巢用1小时1×辛乙封闭溶液在RT，轻微摇动。 </li><li>倒出上清液并更换含二级抗体在适当的稀释比新鲜的1X DIG-B封闭液。染色在RT或O / N卵巢4小时，在4℃，轻微摇动。 <ol><li>使用的第二抗体的以下稀释比:（1）对于免疫荧光染色:1:500的Alexa Fluor 633山羊抗兔IgG或的Al​​exa Fluor 488山羊抗小鼠IgG; （2）为显色染色:1:200，用于生物素化的山羊抗兔IgG。 注:生物素化的第二抗体被施加用于与抗生物素蛋白 - 生物素复合物（ABC）在衬底基（生色）检测，通过该染色信号显著扩增相互作用。 </li><li>对于免疫荧光染色，进行染色在黑暗因为二次抗体是对光敏感。 </li></ol></li><li>用0.2％PBST清洗，每次10分钟洗掉第二抗体的4倍。 </li></ol> 6.核和F-actin的染色注:这仅适用于免疫荧光染色。 <ol><li>卵巢染色用0.2％PBST含DAPI（2纳克/微升）和鬼笔环肽TRITC（100纳米）2小时在室温在黑暗。 </li><li>用0.2％PBST清洗，每次10分钟洗卵巢4次。 </li><li>孵育卵巢与安装介质O / N，在4℃，轻微摇动。 </li></ol> 7.信号开发注:这仅适用于色染色。 <ol><li>制备100微升试剂抗生物素蛋白生物素复合物（ABC）通过在98微升0.2％的PBST加1微升试剂A（抗生物素蛋白）中，通过轻柔的移液充分混合，并加入1微升试剂B的增强信号（生物素与辣根过氧化物酶），接着立即混合。对于在室温30分钟，轻微摇动孵育混合物。 </li><li>孵育卵巢（包括解除关联蛋腔室）中的试剂A和B的混合物在室温30分钟，轻微摇动。 </li><li>用0.2％PBST清洗，每次10分钟洗去试剂A和B的混合物的4倍。 </li><li>转移卵巢淹没在PBST一个很好当场板上用塑料滴管。 </li><li>制备的3,3&#39;-二氨基联苯胺（DAB）底物溶液:溶于1的DAB片剂加另一种含过氧化氢 ​​脲在1ml DDH 2 O的经剧烈涡旋1分钟。 注:DAB，过氧化物酶的底物沉淀，是一种流行的发色体的免疫染色。它产生一个BR被氧化的过氧化物酶后自己的和不溶性沉淀物。弱信号可以通过添加氯化镍至底物溶液（终浓度0.05-0.08％）得到增强。 </li><li>除去残留在阱的PBST溶液和填充用100μl的信号发展的DAB底物溶液。 </li><li>监控信号强度的立体显微镜在低倍镜下下。 </li><li>通过除去DAB溶液，随后用1×PBS立即再填充停止反应。重复洗两次。 </li><li>转移卵巢回1.5 ml的试管中，然后用1×PBS或PBST洗两次，每次15分钟。 </li><li>孵育在基于甘油的封固剂（70％甘油）中O / N的卵巢，在4℃，轻微摇动。 </li></ol> 8.安装蚜虫胚胎注:蚜虫胚胎的厚度发展阶段之间变化。因此，安装策略修改，以适应早期（germaria和阶段0-10），中（阶段11月18日），和晚期胚胎（阶段19-20），其演示了在图2B - ð。胚胎分段接着Miura等。12 <ol><li>立体显微镜下转移卵巢连同安装介质小区托盘用塑料滴管，观察样品在低倍。 </li><li>切割使用昆虫针的侧输卵管相关盏，然后一个孤立卵巢管用滴管转移到一个空的玻璃很好。补足终体积为50-100微升使用的安装介质。 </li><li>转移的卵巢管到幻灯片用玻璃滴管，然后解剖用昆虫针蛋室。 注:对于早于发展阶段6胚胎，蛋室隔离建议;为germaria和前两个蛋商会年龄小于6级，分离是可选的。 </li><li>重新定位一个解剖卵室用玻璃滴管一个干净的幻灯片。 </li><li>把盖玻片（尺寸:22×22MM）在解剖germaria还是先有蛋室（含胚胎阶段1-10的发展）慢慢来避免气泡。 <ol><li>安装蛋室（含有胚胎发育阶段11-18）上的滑动与片面盖玻片桥和放置另一个盖玻片（尺寸:18×18毫米）上的样品的顶部。 </li><li>安装蛋室（含有超过发展阶段19胚胎）上的滑动带双面盖玻片桥和放置另一个盖玻片（尺寸:18×18毫米）上的样品的顶部。 </li></ol></li><li>填充料顶部盖玻片下方的空间安装媒体，以避免干燥的样品。 </li><li>轻度滑动盖玻片，以获得正确的方向观察滚胚胎。 </li><li>密封围盖玻片与指甲油的边缘（包括桥盖玻片）。 </li></ol> 9.成像分析<ol><li>照片微分干涉对比（DIC）的图像整个安装胚胎与compou第二显微镜配备DIC的光学和干物镜（10X，20X，40X），连接到一个摄像机。安装软件使用制造商的说明相机与计算机之间的图像传输。 </li><li>获得用激光扫描共聚焦显微镜14的荧光标记的胚胎的突起。遵循制造商的说明用于拍摄，z轴堆叠，和3D凸出用图像处理软件。 <ol><li>打开滑盖倒置，发现有10倍的目标安装的样品，并圈出样品区，配有油基细笔。 注:这使得识别通过共聚焦显微镜的目标，寻找它时，样品容易。 </li><li>添加一滴油盖玻片上区域，其相反侧作为在9.2.1中描述标记的顶部。 </li></ol></li><li>寻找在40X油浸物镜焦平面然后换一个63X油浸物镜。手动移动精细调焦控制起来，做WN捕捉到最佳焦平面。 注:为了观察germaria和早期胚胎的结构细节，我们建议您使用40倍的目标还是那些具有更高的放大倍率。 </li><li>扫描在不同的激发通道胚胎和获得的Z堆栈图像。 注:对于豌豆蚜组织，降低每个光学部的厚度降低到1.5微米或更小。 </li></ol>

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Cell Biol. 36, 685-695 (1991).</li><li>Wolff, C., Sommer, R., Schroder, R., Glaser, G., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+divergent+expression+aspects+of+the+Drosophila+segmentation+gene+hunchback+in+the+short+germ+band+embryo+of+the+flour+beetle+Tribolium.">Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium.</a> Development. 121 (12), 4227-4236 (1995).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23 (4), 345-358 (2001).</li><li>Lin, G. W., Cook, C. E., Miura, T., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+localization+of+ApVas1+positions+the+preformed+germ+plasm+in+the+sexual+oviparous+pea+aphid+Acyrthosiphon+pisum.">Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum.</a> Evodevo. 5, 18 (2014).</li><li>Straus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+peroxidase+by+methanol+and+by+methanol-nitroferricyanide+for+use+in+immunoperoxidase+procedures.">Inhibition of peroxidase by methanol and by methanol-nitroferricyanide for use in immunoperoxidase procedures.</a> J. Histochem. Cytochem. 19 (11), 682-688 (1971).</li></ol>

The authors have nothing to disclose.

我们确定了最佳条件，以成功的免疫染色豌豆蚜虫的一个关键 ， 豌豆 ，一个新兴的模式生物基因组和发育研究3,15。用于增加组织渗透性和减少的背景染色优化条件下增强的强度和信号的特异性。他们从标准协议有所不同免疫染色在其他动物模型中的步骤创建于细胞膜毛孔和阻断非特异性抗体结合。 用于免疫染色和原位杂交，蛋白酶K（PK）消化?...

蚜虫是半翅目昆虫的小（1-10毫米）的软组织。它们以植物为食吸吮韧皮部汁液用口器刺入。此外，他们依靠专性共生细菌，Buchnera aphidicola，合成的必需氨基酸有缺陷的韧皮部汁液饮食。蚜虫有一个复杂的生活史包括春季和夏季长日照光周期和性卵生复制由短日照的光周期期间，他们躺在越冬卵1,2-有限数量的触发期间孤雌胎生再现。春天，这些卵孵化，生产的第一代全女性蚜虫（fundatrices），继多轮孤雌生殖，直到秋天。周期性孤雌生殖蚜虫，凡在每年的生命周期的无性和有性阶段的交替，已被视为一种进化新奇1,2。在孤雌胎生蚜，胚胎发生在卵巢小管（卵巢管）蛋室的地方。相比之下，性卵生胚胎发育的受精卵。除了生殖可塑性，蚜虫可以显示跨代翼多型:针对拥挤信号和捕食者的威胁，在unwinged无性女性可以viviparously产生翼的后代进行远距离迁移。出版豌豆蚜虫的基因组序列的豌豆蚜 -the的第一基因组序列基底hemimetabolous昆虫可以进一步探索生殖可塑性，翼多型，以及其他功能，包括昆虫与植物相互作用，病毒引导和共生的蚜虫在分子的依据3。 除了 ​​基因组测序，需要用于促进豌豆蚜作为一个成熟的模式生物4用于表征基因表达和功能的工具。我们已经描述了整个底座的可靠的协议<em&gt;在用于蚜虫胚胎5-7检测mRNA的表达原位杂交。通过双链RNA注射和摄食RNA干扰（RNAi）已被用于在蚜虫若虫和成虫的基因沉默，但对于基因抑制在胚胎稳定的条件还没有被报道8-10。免疫染色，可以检测蛋白表达的样品和RNAi敲低后，已对豌豆蚜胚胎11月13日被执行之前的抗体为基础的方法。然而，组织渗透性和消除背景染色增加是因为效果不够理想使用标准协议的免疫染色豌豆蚜虫的无性胎生胚胎。例如，我们发现，组织该抗体的渗透在gastrulating胚胎降低（阶段8-10）和胚胎形态学识别肢芽（阶段13-14）是勉强可渗透抗体。此外，背景染色是可视化的无性viviparo我们豌豆蚜胚胎使用抗体对种系标记分子Vasa以及，对表示在胚胎段12,13的ENGRAILED / Invected蛋白染色。实际上背景染色还在胚胎染色单独用二抗清晰可见。 为了提高导磁率，而不会损坏蚜组织完整性，我们仔细滴定蛋白酶K的浓度，并确定为对蚜虫的胚胎组织消化的最佳条件。为了避免在豌豆蚜非特异性染色，我们搜​​寻了的化合物，可以有效地阻止胚胎和抑制内源过氧化物酶（POD）的活性，用于免疫染色过程中放大信号的酶。通过地高辛（DIG）系缓冲器集提供的，而在传统上使用的正常山羊血清（NGS）甲封闭试剂/牛血清白蛋白（BSA），显著降低背景染色。此外，甲醇，发现INHI更有效地咬了内源性过氧化物酶活性比过氧化氢 ​​（H 2 O 2）。关于这些蚜虫特定条件免疫染色对胚胎的细节将在下面的章节进行说明。

<table><tbody><tr><td>30% hydrogen perxidase (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>18304</td><td>For bleaching endogenous peroxidase of embryos.</td></tr><tr><td>4&amp;rsquo;6-diamidino-2-phenyl-indole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>For labeling the double-strand DNA. TOXIC. Wear gloves, dispense into small aliquots and feeze to minimize exposure</td></tr><tr><td>4D9 anti-engrailed/invected mouse monoclonal antibody</td><td>Developmental studies hybridoma bank</td><td>AB_528224</td><td>For labeling the developing segments of embryo.</td></tr><tr><td>ApVas1 rabbit polyclonal antibody</td><td>Our laboratory</td><td>N/A</td><td>For labeling the aphid germline specific Vas1 protein in embryos. This antibody was made by our laboratory.&amp;nbsp;</td></tr><tr><td>Austerlitz Insect pins</td><td>ENTOMORAVIA</td><td>N/A</td><td>For seperating each egg chambers from an ovariole. Size 000, BLACK ENAMELLED.&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>9048-46-8</td><td>For blocking the non-specific binding of antibodies.</td></tr><tr><td>Camera connted to compound microscope</td><td>Canon</td><td>EOS 5D</td><td>For photographing of aphid embryos.</td></tr><tr><td>Colorimetric 8 cell tray</td><td>Kartell Labware</td><td>357</td><td>For developing the staining signal of aphid embryos. Diameter 95 x 57 mm, cell depth 2 mm.</td></tr><tr><td>Compound microscope with DIC optics</td><td>Leica Microsystems</td><td>DMR</td><td>For photographing of aphid embryo mounted on the slides.</td></tr><tr><td>DIG Wash and Block Buffer Set</td><td>Sigma-Aldrich (Roche)</td><td>11585762001</td><td>For blocking the non-specific binding of antibodies. We diluted the 10x Blocking solution into 1x as blocking reagent.</td></tr><tr><td>Forceps</td><td>Ideal-tek</td><td>N/A</td><td>For dissection of ovaries from aphids. Manufacturer part No 5: 5 SA.&amp;nbsp;</td></tr><tr><td>Glass dropper</td><td>N/A</td><td>N/A</td><td>For transfering ovaries of aphids from the splot plate to tubes. 150 mm of total length and 5 3/4&quot; of tip length.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>For clearing of aphid embryos and mounting.</td></tr><tr><td>Glycine</td><td>Bioshop</td><td>N/A</td><td>For blocking the enzyme activity of proteinase K (PK).</td></tr><tr><td>Goat anti-mouse IgG conjugated Alexa Fluor 488</td><td>Invitrogen</td><td>A11017</td><td>For detection of primary antibody from mouse.</td></tr><tr><td>Goat anti-rabbit IgG conjugated Alexa Fluor 633</td><td>Invitrogen</td><td>A21072</td><td>For detection of primary antibody from rabbit.</td></tr><tr><td>Intelli mixer</td><td>ELMI laboratory equipment</td><td>RM-2M</td><td>For improving the thorughly reaction of reagents with ovaries.</td></tr><tr><td>Laser-scanning confocal microscopy</td><td>Leica Microsystems</td><td>SP5</td><td>For photographing of aphid embryo mounted on the slides with florecent tag.</td></tr><tr><td>methanol&amp;nbsp;</td><td>Burdick and Jackson</td><td>AH2304</td><td>For bleaching endogenous peroxidase of embryos.</td></tr><tr><td>Microscope slides&amp;nbsp;</td><td>Thermo scientific</td><td>10143560</td><td>For mounting of embryos. SUPERFROST ground edges, ca./env. 76 x 26 mm.</td></tr><tr><td>Microscope Cover glasses</td><td>Marienfeld</td><td>0101030</td><td>For mounting embryos on the slides. Size 18 x 18 mm. Thickness No. 1 (0.13 to 0.16 mm)</td></tr><tr><td>Microscope Cover glasses</td><td>Marienfeld</td><td>0101050</td><td>For mounting embryos on the slides. Size 22 x 22 mm. Thickness No. 1 (0.13 to 0.16 mm)</td></tr><tr><td>mouse monoclonal anti-alphaTubulin antibody (DM1A)</td><td>Santa Cruz Biotechnology</td><td>sc-32293</td><td>For labeling the distrbution of alpha-tubulin of cells.</td></tr><tr><td>Nail polish</td><td>N/A</td><td>N/A</td><td>For sealing the coverslips on the slides.</td></tr><tr><td>Normal goat serum (NGS)</td><td>Sigma-Aldrich</td><td>G9023</td><td>For blocking the non-specific binding of antibodies.</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>VWR</td><td>MK262159</td><td>For fixation of aphid ovaries.</td></tr><tr><td>Phalloidin-TRITC</td><td>Sigma-Aldrich</td><td>P1951</td><td>For labeling the distrbution of F-actin on embryos.</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>N/A</td><td>N/A</td><td>As isotonic solution for aphid ovaries.</td></tr><tr><td>Plastic dropper</td><td>N/A</td><td>N/A</td><td>For transfering ovaries of aphids from tube to cell tray. Size 3 ml.&amp;nbsp;</td></tr><tr><td>Proteinase K</td><td>Merck</td><td>124678</td><td>For creating pores (punching) on the cell membrane and facilitating the access of antibodies.&amp;nbsp;</td></tr><tr><td>Pyrex spot plate</td><td>N/A</td><td>N/A</td><td>For dissection and color reactions of aphid ovaries under microscopy. Each cavity with diameter 22 mm and&amp;nbsp; depth 7 mm.</td></tr><tr><td>SIGMAFAST 3,3&amp;rsquo;-diaminobenzidine (DAB) tablets&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D4168</td><td>For developing of substrated signals. Also called DAB peroxidase substrate tablet set.</td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>EZ4</td><td>For dissection and observation of aphid ovaries.</td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td>For creating pores (punching) on the cell membrane.&amp;nbsp;</td></tr><tr><td>VECTASHIELD Elite ABC Kit (Rabbit IgG)</td><td>Vector laboratories</td><td>PK-6101</td><td>For enhancement of the signal. Including of biotinylated goat anti-rabbit IgG secondary antibody, A and B reagents.</td></tr><tr><td>VECTASHIELD mounting medium</td><td>Vector laboratories</td><td>H1000</td><td>For clearing of aphid embryos and mounting.</td></tr></tbody></table>

identification of critical conditions for immunostaining in the pea aphid embryos: increasing tissue permeability and decreasing background staining

在豌豆蚜蚜豌豆 ，用基因组测序和丰富的可塑性，已成为一个新兴的模式，基因组和发育研究。像其他蚜虫，A。通过豌豆孤雌胎生繁殖，其中的胚胎在流水作业的方式，在卵巢管内的卵商会发展迅速传播。此前我们已经建立了全安装了一个强大的平台， 原位杂交技术 ，允许在蚜虫胚胎检测mRNA的表达。分析蛋白质的表达，但是，建立的方案进行免疫染色的无性胎生蚜虫的卵巢管没有产生令人满意的结果。这里，我们报告用于增加组织渗透性和减少的背景染色，这两者都施加既定方法时一些问题的优化条件。优化包括:（1）温育的蛋白酶K（1微克/毫升，10分钟），发现其中必不可少˚F在中期和后期蚜虫胚胎或抗体的渗透; （2）替代正常山羊血清/牛血清白蛋白与由地高辛（DIG）供给的封闭试剂的基于缓冲设置和（3）应用的甲醇，而过氧化氢​​（H 2 O 2）的漂白内源性过氧化物酶;其中显著减少了背景染色的蚜虫组织。对于免疫优化这些关键条件，将允许有效的检测基因产物中的一个， 豌豆等蚜虫胚胎。

在这项研究中，我们进行的无性豌豆蚜虫（ 图1A）的胚胎全安装免疫。这些女性产生后代孤雌及viviparously。这些女性胚胎内的卵巢小管（卵巢管）（ 图1B和 图2A）的蛋商会发展。显微镜之前，​​解剖卵巢管是染色的目标;然而，需要用显微镜（图2B - D）的下观察胚胎的卵腔室分离。 <p class="jove_content" fo:kee...

Watch this Scientific Journal Video about Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining at JoVE.c

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

临界条件在免疫染色豌豆蚜虫胚胎鉴定:增加组织通透性和降低背景染色

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

identification-critical-conditions-for-immunostaining-pea-aphid

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  National Taiwan University. A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

视频: Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

临界条件在免疫染色豌豆蚜虫胚胎鉴定:增加组织通透性和降低背景染色

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蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

整装在鸡的抗体染色方法

光片为主的生活的荧光显微镜或固定和染色

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

阿斯蒂亚纳克斯胚胎的全原位杂交

斑马鱼胚胎和幼虫的整个山免疫性化学

全山原位杂交E8.5，E11.5小鼠胚胎

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法

的透的一种方法