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
Summary
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.
Abstract
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。
Introduction
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>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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 30% hydrogen perxidase (H₂O₂) | Sigma-Aldrich | 18304 | For bleaching endogenous peroxidase of embryos. |
| 4’6-diamidino-2-phenyl-indole dihydrochloride (DAPI) | Sigma-Aldrich | D9542 | For labeling the double-strand DNA. TOXIC. Wear gloves, dispense into small aliquots and feeze to minimize exposure |
| 4D9 anti-engrailed/invected mouse monoclonal antibody | Developmental studies hybridoma bank | AB_528224 | For labeling the developing segments of embryo. |
| ApVas1 rabbit polyclonal antibody | Our laboratory | N/A | For labeling the aphid germline specific Vas1 protein in embryos. This antibody was made by our laboratory. |
| Austerlitz Insect pins | ENTOMORAVIA | N/A | For seperating each egg chambers from an ovariole. Size 000, BLACK ENAMELLED. |
| Bovine serum albumin (BSA) | Sigma-Aldrich | 9048-46-8 | For blocking the non-specific binding of antibodies. |
| Camera connted to compound microscope | Canon | EOS 5D | For photographing of aphid embryos. |
| Colorimetric 8 cell tray | Kartell Labware | 357 | For developing the staining signal of aphid embryos. Diameter 95 x 57 mm, cell depth 2 mm. |
| Compound microscope with DIC optics | Leica Microsystems | DMR | For photographing of aphid embryo mounted on the slides. |
| DIG Wash and Block Buffer Set | Sigma-Aldrich (Roche) | 1.1586E+10 | For blocking the non-specific binding of antibodies. We diluted the 10X Blocking solution into 1X as blocking reagent. |
| Forceps | Ideal-tek | N/A | For dissection of ovaries from aphids. Manufacturer part No 5: 5 SA. |
| Glass dropper | N/A | N/A | For transfering ovaries of aphids from the splot plate to tubes. 150 mm of total length and 5 3/4" of tip length. |
| Glycerol | Sigma-Aldrich | G5516 | For clearing of aphid embryos and mounting. |
| Glycine | Bioshop | N/A | For blocking the enzyme activity of proteinase K (PK). |
| Goat anti-mouse IgG conjugated Alexa Fluor 488 | Invitrogen | A11017 | For detection of primary antibody from mouse. |
| Goat anti-rabbit IgG conjugated Alexa Fluor 633 | Invitrogen | A21072 | For detection of primary antibody from rabbit. |
| Intelli mixer | ELMI laboratory equipment | RM-2M | For improving the thorughly reaction of reagents with ovaries. |
| Laser-scanning confocal microscopy | Leica Microsystems | SP5 | For photographing of aphid embryo mounted on the slides with florecent tag. |
| methanol | Burdick and Jackson | AH2304 | For bleaching endogenous peroxidase of embryos. |
| Microscope slides | Thermo scientific | 10143560 | For mounting of embryos. SUPERFROST ground edges, ca./env. 76 x 26 mm. |
| Microscope Cover glasses | Marienfeld | 0101030 | For mounting embryos on the slides. Size 18 x 18 mm. Thickness No. 1 (0.13 to 0.16 mm) |
| Microscope Cover glasses | Marienfeld | 0101050 | For mounting embryos on the slides. Size 22 x 22 mm. Thickness No. 1 (0.13 to 0.16 mm) |
| mouse monoclonal anti-alphaTubulin antibody (DM1A) | Santa Cruz Biotechnology | sc-32293 | For labeling the distrbution of alpha-tubulin of cells. |
| Nail polish | N/A | N/A | For sealing the coverslips on the slides. |
| Normal goat serum (NGS) | Sigma-Aldrich | G9023 | For blocking the non-specific binding of antibodies. |
| Paraformaldehyde (PFA) | VWR | MK262159 | For fixation of aphid ovaries. |
| Phalloidin-TRITC | Sigma-Aldrich | P1951 | For labeling the distrbution of F-actin on embryos. |
| Phosphate-buffered saline (PBS) | N/A | N/A | As isotonic solution for aphid ovaries. |
| Plastic dropper | N/A | N/A | For transfering ovaries of aphids from tube to cell tray. Size 3 ml. |
| Proteinase K | Merck | 124678 | For creating pores (punching) on the cell membrane and facilitating the access of antibodies. |
| Pyrex spot plate | N/A | N/A | For dissection and color reactions of aphid ovaries under microscopy. Each cavity with diameter 22 mm and depth 7 mm. |
| SIGMAFAST 3,3’-diaminobenzidine (DAB) tablets | Sigma-Aldrich | D4168 | For developing of substrated signals. Also called DAB peroxidase substrate tablet set. |
| Stereo microscope | Leica Microsystems | EZ4 | For dissection and observation of aphid ovaries. |
| Triton-X 100 | Sigma-Aldrich | T8787 | For creating pores (punching) on the cell membrane. |
| VECTASHIELD Elite ABC Kit (Rabbit IgG) | Vector laboratories | PK-6101 | For enhancement of the signal. Including of biotinylated goat anti-rabbit IgG secondary antibody, A and B reagents. |
| VECTASHIELD mounting medium | Vector laboratories | H1000 | For clearing of aphid embryos and mounting. |
Riferimenti
- Blackman, R. L., Minks, A. K., Harrewijn, P. Reproduction, cytogenetics and development. Aphids: their biology, natural enemies. , 163-195 (1987).
- Davis, G. K. Cyclical parthenogenesis and viviparity in aphids as evolutionary novelties. J. Exp. Zool. 318, 448-459 (2012).
- The International Aphid Genomics Consortium. Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biol. 8 (2), e1000313 (2010).
- Abzhanov, A., et al. Are we there yet? Tracking the development of new model systems. Trends Genet. 24 (7), 353-360 (2008).
- Chang, C., et al. Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea)). Dev. Genes Evol. 217 (4), 275-287 (2007).
- Chang, C., et al. Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility. Arch. Insect Biochem. 68 (4), 186-196 (2008).
- Chung, C. Y., Cook, C. E., Lin, G. W., Huang, T. Y., Chang, C. Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis. Insect Sci. 21 (3), 265-277 (2014).
- Mutti, N. S., Park, Y., Reese, J. C., Reeck, G. R. RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum. J. Insect Sci. 6, 38 (2006).
- Jaubert-Possamai, S., et al. Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum. BMC Biotechnol. 7, 63 (2007).
- Sapountzis, P., et al. New insight into the RNA interference response against cathepsin-L gene in the pea aphid, Acyrthosiphon pisum: Molting or gut phenotypes specifically induced by injection or feeding treatments. Insect Biochem. Mol. Biol. 51, 20-32 (2014).
- Braendle, C., et al. Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis. PLoS Biol. 1 (1), e21 (2003).
- Miura, T., et al. A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). J. Exp. Zool. 295 (1), 59-81 (2003).
- Chang, C., Lee, W. C., Cook, C. E., Lin, G. W., Chang, T. Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers. Int. J. Dev. Biol. 50 (4), 413-421 (2006).
- Koshy, A. A., Cabral, C. M. 3-D imaging and analysis of neurons infected in vivo with Toxoplasma gondii. J. Vis. Exp. (94), e52237 (2014).
- Brisson, J. A., Stern, D. L. The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies. Bioessays. 28 (7), 747-755 (2006).
- Tautz, D., Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 98 (2), 81-85 (1989).
- Harland, R. M. In-situ hybridization: an improved whole-mount method for Xenopus embryos. Method. Cell Biol. 36, 685-695 (1991).
- Wolff, C., Sommer, R., Schroder, R., Glaser, G., Tautz, D. Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium. Development. 121 (12), 4227-4236 (1995).
- Jowett, T. Double in situ hybridization techniques in zebrafish. Methods. 23 (4), 345-358 (2001).
- Lin, G. W., Cook, C. E., Miura, T., Chang, C. Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum. Evodevo. 5, 18 (2014).
- Straus, W. Inhibition of peroxidase by methanol and by methanol-nitroferricyanide for use in immunoperoxidase procedures. J. Histochem. Cytochem. 19 (11), 682-688 (1971).
