Oogenesis pp 19-33 | Cite as

Antibody Staining in Drosophila Germaria

  • Anette Lie-Jensen
  • Kaisa HaglundEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1457)


Drosophila oogenesis is a powerful model for studying a wide spectrum of cellular and developmental processes in vivo. Oogenesis starts in a specialized structure called the germarium, which harbors the stem cells for both germ and somatic cells. The germarium produces egg chambers, each of which will develop into an egg. Active areas of research in Drosophila germaria include stem cell self-renewal, division, and maintenance, cell cycle control and differentiation, oocyte specification, intercellular communication, and signaling, among others. The solid knowledge base, the genetic tractability of the Drosophila model, as well as the availability and fast development of tools and imaging techniques for oogenesis research ensure that studies in this model will keep being instrumental for novel discoveries within cell and developmental biology also in the future. This chapter focuses on antibody staining in Drosophila germaria and provides a protocol for immunostaining as well as an overview of commonly used antibodies for visualization of different cell types and cellular structures. The protocol is well-suited for subsequent confocal microscopy analyses, and in addition we present key adaptations of the protocol that are useful when performing structured illumination microscopy (SIM) super-resolution imaging.

Key words

Drosophila oogenesis Germarium Germline stem cell Germline cyst Fusome Ring canal Follicle cell Antibody staining Structured illumination microscopy 



We thank Vigdis Sørensen for assistance with 3D SIM super-resolution imaging, David Glover for providing the w1118; p[w + Ub-GFP-Pav-KLP]53 fly line, and the Developmental Studies Hybridoma Bank (DSHB) for antibodies. K.H acknowledges a career researcher grant from the South-Eastern Norway Regional Health Authority, project number 2012054. A.L-J is supported by this grant. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179571. The confocal microscopy core facility at Oslo University Hospital is acknowledged for access to confocal microscopes. The super-resolution microscopy core facility at Oslo University Hospital is acknowledged for access to a DeltaVision OMX microscope.


  1. 1.
    Fuller MT, Spradling AC (2007) Male and female Drosophila germline stem cells: two versions of immortality. Science 316(5823):402–404. doi: 10.1126/science.1140861 CrossRefPubMedGoogle Scholar
  2. 2.
    Spradling A, Fuller MT, Braun RE, Yoshida S (2011) Germline stem cells. Cold Spring Harb Perspect Biol 3(11):a002642. doi: 10.1101/cshperspect.a002642 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Eliazer S, Buszczak M (2011) Finding a niche: studies from the Drosophila ovary. Stem Cell Res Ther 2(6):45. doi: 10.1186/scrt86 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Losick VP, Morris LX, Fox DT, Spradling A (2011) Drosophila stem cell niches: a decade of discovery suggests a unified view of stem cell regulation. Dev Cell 21(1):159–171. doi: 10.1016/j.devcel.2011.06.018 CrossRefPubMedGoogle Scholar
  5. 5.
    Yan D, Neumuller RA, Buckner M, Ayers K, Li H, Hu Y, Yang-Zhou D, Pan L, Wang X, Kelley C, Vinayagam A, Binari R, Randklev S, Perkins LA, Xie T, Cooley L, Perrimon N (2014) A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell 28(4):459–473. doi: 10.1016/j.devcel.2014.01.020 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    de Cuevas M, Spradling AC (1998) Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125(15):2781–2789PubMedGoogle Scholar
  7. 7.
    Mathieu J, Cauvin C, Moch C, Radford SJ, Sampaio P, Perdigoto CN, Schweisguth F, Bardin AJ, Sunkel CE, McKim K, Echard A, Huynh JR (2013) Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells. Dev Cell 26(3):250–265. doi: 10.1016/j.devcel.2013.07.005 CrossRefPubMedGoogle Scholar
  8. 8.
    Salzmann V, Chen C, Chiang CY, Tiyaboonchai A, Mayer M, Yamashita YM (2014) Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division. Mol Biol Cell 25(2):267–275. doi: 10.1091/mbc.E13-09-0541 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Eikenes AH, Malerod L, Christensen AL, Steen CB, Mathieu J, Nezis IP, Liestol K, Huynh JR, Stenmark H, Haglund K (2015) ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo. PLoS Genet 11(1):e1004904. doi: 10.1371/journal.pgen.1004904 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Matias NR, Mathieu J, Huynh JR (2015) Abscission is regulated by the ESCRT-III protein shrub in Drosophila germline stem cells. PLoS Genet 11(2):e1004653. doi: 10.1371/journal.pgen.1004653 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ables ET, Drummond-Barbosa D (2013) Cyclin E controls Drosophila female germline stem cell maintenance independently of its role in proliferation by modulating responsiveness to niche signals. Development 140(3):530–540. doi: 10.1242/dev.088583 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Pepling ME, de Cuevas M, Spradling AC (1999) Germline cysts: a conserved phase of germ cell development? Trends Cell Biol 9(7):257–262CrossRefPubMedGoogle Scholar
  13. 13.
    Lilly MA, de Cuevas M, Spradling AC (2000) Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary. Dev Biol 218(1):53–63. doi: 10.1006/dbio.1999.9570 CrossRefPubMedGoogle Scholar
  14. 14.
    Hawkins NC, Thorpe J, Schupbach T (1996) Encore, a gene required for the regulation of germ line mitosis and oocyte differentiation during Drosophila oogenesis. Development 122(1):281–290PubMedGoogle Scholar
  15. 15.
    Tastan OY, Maines JZ, Li Y, McKearin DM, Buszczak M (2010) Drosophila ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts. Development 137(19):3167–3176. doi: 10.1242/dev.050575 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    McKearin DM, Spradling AC (1990) bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis. Genes Dev 4(12B):2242–2251CrossRefPubMedGoogle Scholar
  17. 17.
    Huynh JR, St Johnston D (2004) The origin of asymmetry: early polarisation of the Drosophila germline cyst and oocyte. Curr Biol 14(11):R438–R449. doi: 10.1016/j.cub.2004.05.040 CrossRefPubMedGoogle Scholar
  18. 18.
    Bastock R, St Johnston D (2008) Drosophila oogenesis. Curr Biol 18(23):R1082–R1087. doi: 10.1016/j.cub.2008.09.011 CrossRefPubMedGoogle Scholar
  19. 19.
    Lake CM, Hawley RS (2012) The molecular control of meiotic chromosomal behavior: events in early meiotic prophase in Drosophila oocytes. Annu Rev Physiol 74:425–451. doi: 10.1146/annurev-physiol-020911-153342 CrossRefPubMedGoogle Scholar
  20. 20.
    Christophorou N, Rubin T, Huynh JR (2013) Synaptonemal complex components promote centromere pairing in pre-meiotic germ cells. PLoS Genet 9(12):e1004012. doi: 10.1371/journal.pgen.1004012 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Christophorou N, Rubin T, Bonnet I, Piolot T, Arnaud M, Huynh JR (2015) Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol 17:1388–400. doi: 10.1038/ncb3249 CrossRefPubMedGoogle Scholar
  22. 22.
    Kai T, Spradling A (2004) Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries. Nature 428(6982):564–569. doi: 10.1038/nature02436 CrossRefPubMedGoogle Scholar
  23. 23.
    Hartman TR, Ventresca EM, Hopkins A, Zinshteyn D, Singh T, O’Brien JA, Neubert BC, Hartman MG, Schofield HK, Stavrides KP, Talbot DE, Riggs DJ, Pritchard C, O’Reilly AM (2015) Novel tools for genetic manipulation of follicle stem cells in the Drosophila ovary reveal an integrin-dependent transition from quiescence to proliferation. Genetics 199(4):935–957. doi: 10.1534/genetics.114.173617 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Drummond-Barbosa D, Spradling AC (2001) Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev Biol 231(1):265–278. doi: 10.1006/dbio.2000.0135 CrossRefPubMedGoogle Scholar
  25. 25.
    Chang YC, Jang AC, Lin CH, Montell DJ (2013) Castor is required for Hedgehog-dependent cell-fate specification and follicle stem cell maintenance in Drosophila oogenesis. Proc Natl Acad Sci U S A 110(19):E1734–E1742. doi: 10.1073/pnas.1300725110 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Forbes AJ, Lin H, Ingham PW, Spradling AC (1996) Hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila. Development 122(4):1125–1135PubMedGoogle Scholar
  27. 27.
    Horne-Badovinac S, Bilder D (2005) Mass transit: epithelial morphogenesis in the Drosophila egg chamber. Dev Dyn 232(3):559–574. doi: 10.1002/dvdy.20286 CrossRefPubMedGoogle Scholar
  28. 28.
    McLaughlin JM, Bratu DP (2015) Drosophila melanogaster oogenesis: an overview. Methods Mol Biol 1328:1–20. doi: 10.1007/978-1-4939-2851-4_1 CrossRefPubMedGoogle Scholar
  29. 29.
    Song X, Zhu CH, Doan C, Xie T (2002) Germline stem cells anchored by adherens junctions in the Drosophila ovary niches. Science 296(5574):1855–1857. doi: 10.1126/science.1069871 CrossRefPubMedGoogle Scholar
  30. 30.
    Lopez-Onieva L, Fernandez-Minan A, Gonzalez-Reyes A (2008) Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary. Development 135(3):533–540. doi: 10.1242/dev.016121 CrossRefPubMedGoogle Scholar
  31. 31.
    Wang L, Li Z, Cai Y (2008) The JAK/STAT pathway positively regulates DPP signaling in the Drosophila germline stem cell niche. J Cell Biol 180(4):721–728. doi: 10.1083/jcb.200711022 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Chen D, McKearin D (2003) Dpp signaling silences bam transcription directly to establish asymmetric divisions of germline stem cells. Curr Biol 13(20):1786–1791CrossRefPubMedGoogle Scholar
  33. 33.
    Song X, Wong MD, Kawase E, Xi R, Ding BC, McCarthy JJ, Xie T (2004) Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag of marbles, in germline stem cells in the Drosophila ovary. Development 131(6):1353–1364. doi: 10.1242/dev.01026 CrossRefPubMedGoogle Scholar
  34. 34.
    Li Y, Minor NT, Park JK, McKearin DM, Maines JZ (2009) Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance. Proc Natl Acad Sci U S A 106(23):9304–9309. doi: 10.1073/pnas.0901452106 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Robinson DN, Cooley L (1997) Genetic analysis of the actin cytoskeleton in the Drosophila ovary. Annu Rev Cell Dev Biol 13:147–170. doi: 10.1146/annurev.cellbio.13.1.147 CrossRefPubMedGoogle Scholar
  36. 36.
    Lin H, Yue L, Spradling AC (1994) The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation. Development 120(4):947–956PubMedGoogle Scholar
  37. 37.
    Hudson AM, Cooley L (2014) Methods for studying oogenesis. Methods 68(1):207–217. doi: 10.1016/j.ymeth.2014.01.005 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Buszczak M, Paterno S, Lighthouse D, Bachman J, Planck J, Owen S, Skora AD, Nystul TG, Ohlstein B, Allen A, Wilhelm JE, Murphy TD, Levis RW, Matunis E, Srivali N, Hoskins RA, Spradling AC (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175(3):1505–1531. doi: 10.1534/genetics.106.065961 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Quinones-Coello AT, Petrella LN, Ayers K, Melillo A, Mazzalupo S, Hudson AM, Wang S, Castiblanco C, Buszczak M, Hoskins RA, Cooley L (2007) Exploring strategies for protein trapping in Drosophila. Genetics 175(3):1089–1104. doi: 10.1534/genetics.106.065995 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Ni JQ, Zhou R, Czech B, Liu LP, Holderbaum L, Yang-Zhou D, Shim HS, Tao R, Handler D, Karpowicz P, Binari R, Booker M, Brennecke J, Perkins LA, Hannon GJ, Perrimon N (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8(5):405–407. doi: 10.1038/nmeth.1592 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Fichelson P, Moch C, Ivanovitch K, Martin C, Sidor CM, Lepesant JA, Bellaiche Y, Huynh JR (2009) Live-imaging of single stem cells within their niche reveals that a U3snoRNP component segregates asymmetrically and is required for self-renewal in Drosophila. Nat Cell Biol 11(6):685–693. doi: 10.1038/ncb1874 CrossRefPubMedGoogle Scholar
  42. 42.
    Lenhart KF, DiNardo S (2015) Somatic cell encystment promotes abscission in germline stem cells following a regulated block in cytokinesis. Dev Cell 34(2):192–205. doi: 10.1016/j.devcel.2015.05.003 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Sheng XR, Matunis E (2011) Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output. Development 138(16):3367–3376. doi: 10.1242/dev.065797 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Bianco A, Poukkula M, Cliffe A, Mathieu J, Luque CM, Fulga TA, Rorth P (2007) Two distinct modes of guidance signalling during collective migration of border cells. Nature 448(7151):362–365. doi: 10.1038/nature05965 CrossRefPubMedGoogle Scholar
  45. 45.
    Prasad M, Jang AC, Starz-Gaiano M, Melani M, Montell DJ (2007) A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging. Nat Protoc 2(10):2467–2473. doi: 10.1038/nprot.2007.363 CrossRefPubMedGoogle Scholar
  46. 46.
    Gilliland WD, Hughes SE, Cotitta JL, Takeo S, Xiang Y, Hawley RS (2007) The multiple roles of mps1 in Drosophila female meiosis. PLoS Genet 3(7):e113. doi: 10.1371/journal.pgen.0030113 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Morris LX, Spradling AC (2011) Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary. Development 138(11):2207–2215. doi: 10.1242/dev.065508 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    He L, Wang X, Montell DJ (2011) Shining light on Drosophila oogenesis: live imaging of egg development. Curr Opin Genet Dev 21(5):612–619. doi: 10.1016/j.gde.2011.08.011 CrossRefPubMedGoogle Scholar
  49. 49.
    Prasad M, Wang X, He L, Montell DJ (2011) Border cell migration: a model system for live imaging and genetic analysis of collective cell movement. Methods Mol Biol 769:277–286. doi: 10.1007/978-1-61779-207-6_19 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Luo L, Chai PC, Cai Y (2013) Immunostaining of germline stem cells and the niche in Drosophila ovaries. Methods Mol Biol 1035:1–7. doi: 10.1007/978-1-62703-508-8_1 CrossRefPubMedGoogle Scholar
  51. 51.
    Hanyu-Nakamura K, Kobayashi S, Nakamura A (2004) Germ cell-autonomous Wunen2 is required for germline development in Drosophila embryos. Development 131(18):4545–4553. doi: 10.1242/dev.01321 CrossRefPubMedGoogle Scholar
  52. 52.
    Hong A, Lee-Kong S, Iida T, Sugimura I, Lilly MA (2003) The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I. Development 130(7):1235–1242CrossRefPubMedGoogle Scholar
  53. 53.
    Forbes AJ, Spradling AC, Ingham PW, Lin H (1996) The role of segment polarity genes during early oogenesis in Drosophila. Development 122(10):3283–3294PubMedGoogle Scholar
  54. 54.
    Haglund K, Nezis IP, Lemus D, Grabbe C, Wesche J, Liestol K, Dikic I, Palmer R, Stenmark H (2010) Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster. Curr Biol 20(10):944–950. doi: 10.1016/j.cub.2010.03.068 CrossRefPubMedGoogle Scholar
  55. 55.
    Patel NH, Snow PM, Goodman CS (1987) Characterization and cloning of fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. Cell 48(6):975–988CrossRefPubMedGoogle Scholar
  56. 56.
    O’Reilly AM, Ballew AC, Miyazawa B, Stocker H, Hafen E, Simon MA (2006) Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells. Development 133(14):2627–2638. doi: 10.1242/dev.02423 CrossRefPubMedGoogle Scholar
  57. 57.
    Dubreuil R, Byers TJ, Branton D, Goldstein LS, Kiehart DP (1987) Drosophila spectrin. I. Characterization of the purified protein. J Cell Biol 105(5):2095–2102CrossRefPubMedGoogle Scholar
  58. 58.
    Lighthouse DV, Buszczak M, Spradling AC (2008) New components of the Drosophila fusome suggest it plays novel roles in signaling and transport. Dev Biol 317(1):59–71. doi: 10.1016/j.ydbio.2008.02.009 CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Robinson DN, Cant K, Cooley L (1994) Morphogenesis of Drosophila ovarian ring canals. Development 120(7):2015–2025PubMedGoogle Scholar
  60. 60.
    Minestrini G, Mathe E, Glover DM (2002) Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis. J Cell Sci 115(Pt 4):725–736PubMedGoogle Scholar
  61. 61.
    Zhang Y, Kalderon D (2001) Hedgehog acts as a somatic stem cell factor in the Drosophila ovary. Nature 410(6828):599–604. doi: 10.1038/35069099 CrossRefPubMedGoogle Scholar
  62. 62.
    Grieder NC, de Cuevas M, Spradling AC (2000) The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127(19):4253–4264PubMedGoogle Scholar
  63. 63.
    Verheyen EM, Cooley L (1994) Profilin mutations disrupt multiple actin-dependent processes during Drosophila development. Development 120(4):717–728PubMedGoogle Scholar
  64. 64.
    Zimmerman SG, Peters NC, Altaras AE, Berg CA (2013) Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat Protoc 8(11):2158–2179. doi: 10.1038/nprot.2013.136 CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Haack T, Bergstralh DT, St Johnston D (2013) Damage to the Drosophila follicle cell epithelium produces “false clones” with apparent polarity phenotypes. Biol Open 2(12):1313–1320. doi: 10.1242/bio.20134671 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular Cell Biology, Institute for Cancer ResearchOslo University HospitalOsloNorway
  2. 2.Centre for Cancer Biomedicine, Faculty of MedicineUniversity of OsloOsloNorway

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