Immunohistochemistry in Drosophila

Sections and Whole Mounts
  • Charlotte Helfrich-Förster
Part of the Methods in Molecular Biology™ book series (MIMB, volume 362)


This chapter describes immunohistochemistry on Drosophila heads (brains) with respect to antigens involved in the circadian system. Two different methods have been successfully performed in several labs: immunolabeling on whole-mount brains and on cryostat sections of entire heads. Both methods are addressed here. The primary antisera can be detected by enzyme-labeled or fluorescence-labeled secondary antibodies. The advantages of the different methods are discussed.

Key Words

Antigen antiserum brain circadian rhythms compound eyes cryostat sections Drosophila fluorescence period horseradish peroxidase immunohistochemistry peroxidase-antiperoxidase secondary antibody timeless whole mounts 


  1. 1.
    Sternberger, L. A. (1979) Immunocytochemistry. Wiley, New York.Google Scholar
  2. 2.
    Pollak, J. M., and Van Norden, S. (1983) Immunocytochemistry, Practical Applications in Pathology and Biology. Wright, Bristol, UK.Google Scholar
  3. 3.
    Cuello, A. C. (1983) Immunohistochemistry. Wiley, New York.Google Scholar
  4. 4.
    Schoeneveld, H. and Veenstra J. A. (1988) Immunocytochemistry. In: Immunological Techniques in Insect Biology (Gilbert, L. I., and Miller T. A., eds.), Springer, New York, pp. 93–133.Google Scholar
  5. 5.
    White, R. A. H. (1998) Immunolabeling of Drosophila. In: Drosophila, A Practical Approach (Roberts, D. B., ed.), Oxford University Press, Oxford, pp. 215–240.Google Scholar
  6. 6.
    Ashburner, M. (1989) Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  7. 7.
    Stanewsky, R., Frisch, B., Brandes, C., Hamblen-Coyle, M.J., Rosbash, M., and Hall, J. C. (1997) Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling. J. Neurosci. 17, 676–696.PubMedGoogle Scholar
  8. 8.
    Kaneko, M., Helfrich-Förster, C., and Hall, J. C. (1997) Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: newly identified pacemaker candidates and novel features of clock gene product cycling. J. Neurosci. 17, 6745–6760.PubMedGoogle Scholar
  9. 9.
    Kloss, B., Rothenfluh, A., Young, M. W., and Saez, L. (2001) Phosphorylation of Period is influenced by cycling physical associations of Double-time, Period, and Timeless in the Drosophila clock. Neuron 30, 699–706.CrossRefPubMedGoogle Scholar
  10. 10.
    Emery, P., So, W. V., Kaneko, M., Hall, J. C., and Rosbash, M. (1998) CRY, a Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity. Cell 95, 669–679.CrossRefPubMedGoogle Scholar
  11. 11.
    Dircksen, H., Zahnow, G., Gaus, R., Keller, R., Rao, K. R., and Riehm, J. P. (1987) The ultrastructure of nerve endings containing pigment-dispersing homone (PDH) in crustacean sinus glands: identification by an antiserum against synthetic PDH. Cell Tissue Res. 250, 377–387.CrossRefGoogle Scholar
  12. 12.
    Park, J. H., Helfrich-Förster, C., Lee, G., Liu, L., Rosbash, M., and Hall, J. C. (2000) Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc. Nat. Acad. Sci. USA 97, 3608–3613.CrossRefPubMedGoogle Scholar
  13. 13.
    Sternberger, L. A. and Sternberger, N. H. (1986) The unlabelled antibody method: Comparison of peroxidase-antiperoxidase with avidine-biotin complex by a new method of quantification. J. Histochem. Cytochem. 34, 599–605.PubMedGoogle Scholar
  14. 14.
    Renn, S. C. P., Park, J. H., Rosbash, M., Hall J. C., and Taghert, P. H. (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99, 791–802.CrossRefPubMedGoogle Scholar
  15. 15.
    Kaneko, M., and Hall, J. C. (2000) Neuroanatomy of cells expressing clock genes in Drosophila: Transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 422, 66–94.CrossRefPubMedGoogle Scholar
  16. 16.
    Würden, S., and Homberg, U. (1993) A simple method for immunofluorescent double staining with primary antisera from the same species. J. Histochem. Cytochem. 41, 627–630.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Charlotte Helfrich-Förster
    • 1
  1. 1.Institut für ZoologieUniversität RegensburgRegensburgGermany

Personalised recommendations