Links and References

Whole mount in situ hybridization:

• D. Tautz and C. Pfeifle (1989). 'A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback.' Chromosoma 98, 81-85.....This paper introduces whole mount in situ hybridization and immunohistochemical detection of mRNA in Drosophila embryos.
• J. Jiang, D. Kosman, Y.T. Ip, and M. Levine (1991). 'The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos.' Genes and Development 5, 1881-1891.....This paper describes one of the first applications of DIG-labeled RNA probes.
• R. Lehmann and D. Tautz (1994). 'In situ hybridization to RNA.' In: 'Drosophila melanogaster: practical uses in cell and molecular biology', Methods in Cell Biology 44, eds. L.S.B. Goldstein and E.A. Fyrberg, Academic Press, 575-598.....This chapter further describes the basic method and some modifications, including the use of RNA probes.

Immunohistochemical staining methods for multiple mRNAs:

• J.W. O'Neill and E. Bier (1994). 'Double-Label In Situ Hybridization Using Biotin and Digoxigenin-Tagged RNA Probes.' BioTechniques 17, 870-875.....This protocol can be viewed also on this web page. DIG and BIO probes are detected with alkaline phosphatase and horseradish peroxidase, producing a blue and brown double stain.
• D. Kosman and S. Small (1997). 'Concentration-dependent patterning by an ectopic expression domain of the Drosophila gap gene knirps.' Development 124, 1343-1354.....DIG and FITC probes are detected with sequential alkaline phosphatase reactions, producing a red and black double stain.
• G. Hauptmann (2001). 'One-, Two-, and Three-Color Whole-Mount in Situ Hybridization to Drosophila Embryos.' Methods 23, 359-372.....DIG, BIO, and FITC probes are detected with sequential alkaline phosphatase reactions using a variety of substrates, also in combination with the blue reaction product of beta-galactosidase.
• S. Long and M. Rebagliati (2002). 'Sensitive Two-Color Whole-Mount In Situ Hybridizations Using Digoxygenin- and Dinitrophenol-Labeled RNA Probes.' BioTechniques 32, 494-500.....DIG and DNP probes are detected in Xenopus embryos with sequential alkaline phosphatase reactions, using INT RED/BCIP and BM Purple substrates, producing a red and purple double stain.

Single and double fluorescent mRNA in situ protocols:

• N. Kagiyama, K. Yoshida, T. Hamabata, N. Juni, T. Awasaki, S. Fujita, M. Momiyama, Y. Kondoh, M.C. Yoshida and S.H. Hori. (1993). 'A novel fluorescent method for in situ hybridization.' Acta Histochem. Cytochem. 26, 441-445.....The alkaline phosphatase substrate Fast Red is used to produce a red fluorescent mRNA stain.
• S.C. Hughes, B. Saulier-Le Drean, I. Livne-Bar and H.M. Krause (1996). 'Fluorescence In Situ Hybridization in Whole-Mount Drosophila Embryos.' BioTechniques 20: 748-750.....FITC probes are detected with an unconjugated monoclonal antibody followed by a fluorescent secondary.
• S.C. Hughes and H.M. Krause (1998). 'Double Labeling with Fluorescence In Situ Hybridization in Drosophila Whole-Mount Embryos.' BioTechniques 24, 530-532.....FITC and DIG probes are detected with two primary antibodies followed by red and green fluorescent secondary antibodies.
• S.C. Hughes and H.M. Krause (1998). 'Single and double FISH protocols for Drosophila.' In: 'Confocal Microscopy Methods and Protocols', Methods in Molecular Biology 122, ed. S.W. Paddock, Humana Press, 93-101.....This chapter presents the two previous protocols, which can be viewed also on this web page.
• G.S. Wilkie and I. Davis (1998). 'Visualizing mRNA by in situ hybridization using high resolution and sensitive tyramide signal amplification.' Elsevier Trends Journals, Technical Tips Online: T01458.....DIG and FITC probes are visualized using sequential fluorescent tyramide reactions.
• C. Paratore, U. Suter, and L. Sommer (1999). 'Embryonic gene expression resolved at the cellular level by fluorescence in situ hybridization.' Histochemistry and Cell Biology 111, 435-443.....DIG and FITC probes are visualized in mouse embryo sections with sequential tyramide reactions.
• T. Jowett (2001). 'Direct fluorescence in situ hybridization to Drosophila embryos.' Oxford Practical Approach Online, Oxford University Press (originally published in 'In Situ Hybridization- A Practical Approach, second edition', ed. D.G. Wilkinson, 1998.....FITC probes are visualized directly, and DIG probes indirectly with alkaline phosphatase and Fast Red.
• T. Jowett (2001). 'Double fluorescent in situ hybridization of zebrafish or Drosophila embryos.' Oxford Practical Approach Online, Oxford University Press (originally published in 'In Situ Hybridization- A Practical Approach, second edition', ed. D.G. Wilkinson, 1998.....DIG and FITC probes are detected with sequential alkaline phosphatase reactions using the substrates ELF97 and Fast Red.

Methods for simultaneous detection of mRNA and protein:

• M.A. Sturtevant, M. Roark, and E. Bier (1993). 'The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway.' Genes and Development 7, 961-973.....A biotinylated secondary is used to label protein before hybridization, which is then carried out in the standard way, and both protein and mRNA signals are histochemically developed.
• S. Goto and S. Hayashi (1997). 'Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein.' Development, Genes and Evolution 207, 194-198.....A fluorescent secondary is used to label protein before hybridization, and mRNA is visualized with alkaline phosphatase and Fast Red.
• S. Knirr, N. Azpiazu, and M. Frasch (1999). 'The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.' Development 126, 4525-4535.....ProtK treatment is omitted, DIG probes are detected first with fluorescent tyramide, and then protein is labeled with fluorescent secondaries or another tyramide reaction.
• A.U. Zaidi, H. Enomoto, J. Milbrandt, and K.A. Roth (2000). 'Dual Fluorescent In Situ Hybridization and Immunohistochemical Detection with Tyramide Signal Amplification.' Journal of Histochemistry and Cytochemistry 48, 1369-1375.....Protein and mRNA are detected in mouse brain sections with sequential fluorescent tyramide reactions.
• H. Nagaso, T. Murata, N. Day, and K.K. Yokoyama (2001). 'Simultaneous Detection of RNA and Protein by In Situ Hybridization and Immunological Staining.' Journal of Histochemistry and Cytochemistry 49, 1177-1182.....Acetone is used as a substitute for ProtK in order to detect both protein and mRNA in both embryos and wing discs.
• X. Wu, V. Vasisht, D. Kosman, J. Reinitz, and S. Small (2001). 'Thoracic Patterning by the Drosophila Gap Gene hunchback.' Developmental Biology 237, 79-92.....ProtK treatment is omitted, primary and fluorescent secondary antibodies are used to detect proteins post-hybridization, while DIG probes are detected with primary/biotinylated secondary antibodies and fluorescein-avidin.

Nascent transcript M-FISH:

• P.H. O'Farrell, B.A. Edgar, D. Lakich, and C.F. Lehner (1989). 'Directing cell division during development.' Science 246, 635-640.
• A.W. Shermoen and P.H. O'Farrell (1991). 'Progression of the cell cycle through mitosis leads to abortion of nascent transcripts.' Cell 67, 303-310.
• D.K. Pritchard and G. Schubiger (1996). 'Activation of transcription in Drosophila embryos is a gradual process mediated by the nucleocytoplasmic ratio.' Genes and Development 10, 1131-1142.
• M.R. Speicher, S. Gwyn Ballard, and D.C. Ward (1996). 'Karyotyping human chromosomes by combinatorial multi-fluor FISH.' Nature Genetics 12, 368-375.
• G.S. Wilkie, A.W. Shermoen, P.H. O'Farrell, and I. Davis (1999). 'Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei.' Current Biology 9, 1263-1266.
• O. Henegariu, P. Bray-Ward, and D.C. Ward (2000). 'Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling.' Nature Biotechnology 18, 345-348.
• J.M. Levsky, S.M. Shenoy, R.C. Pezo, and R.H. Singer (2002). 'Single-cell gene expression profiling.' Science 297, 836-840.
• J. Seidel, A. Heller, G. Senger, H. Starke, I. Chudoba, C. Kelbova, H. Tonnies, H. Neitzel, C. Haase, V. Beensen, F. Zintl, U. Claussen, and T. Liehr (2003). 'A multiple translocation event in a patient with hexadactyly, facial dysmorphism, mental retardation and behaviour disorder characterised comprehensively by molecular cytogenetics. Case report and review of the literature.' European Journal of Pediatrics online publication, June 19 2003.
• Numerous online sources of M-FISH information can be found. For example, see this web page by John Miklos for a summary of several techniques, or Tavi's Multicolor FISH page by Octavian Henegariu for detailed protocols.

Fluorescence imaging techniques:

• E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Ried (1996). 'Multicolor spectral karyotyping of human chromosomes.' Science 273, 494-497.
• A.M. Femino, F.S. Fay, K. Fogarty, and R.H. Singer (1998). 'Visualization of single RNA transcripts in situ.' Science 280, 585-590.
• C. Ortiz de Solorzano, E. Garcia Rodriguez, A. Jones, D. Pinkel, J.W. Gray, D. Sudar, and S.J. Lockett (1999). 'Segmentation of confocal microscope images of cell nuclei in thick tissue sections.' Journal of Microscopy 193, 212-226.
• H. Tsurui, H. Nishimura, S. Hattori, S. Hirose, K. Okumura, and T. Shirai (2000). 'Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.' Journal of Histochemistry and Cytochemistry 48, 653-662.
• C. Ortiz de Solorzano, R. Malladi, S.A. Lelievre, and S.J. Lockett (2001). 'Segmentation of nuclei and cells using membrane related protein markers.' Journal of Microscopy 201, 404-415.
• S. Paddock (2001). 'Channel surfing: creating different color combinations from multiple-label images.' BioTechniques 30, 756-761.
• M.E. Dickinson, G. Bearman, S. Tille, R. Lansford, and S.E. Fraser (2001). 'Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy.' BioTechniques 31, 1272-1278.

Gene expression projects:

• J. Rusch and M. Levine (1996). 'Threshold responses to the dorsal regulatory gradient and the subdivision of primary tissue territories in the Drosophila embryo.' Current Opinion in Genetics and Development 6, 416-423.
• E. Myasnikova, A. Samsonova, K. Kozlov, M. Samsonova, and J. Reinitz (2001). 'Registration of the expression patterns of Drosophila segmentation genes by two independent methods.' Bioinformatics 17, 3-12.
• K. Kozlov, E. Myasnikova, A. Pisarev, M. Samsonova, and J. Reinitz (2002). 'A method for two-dimensional registration and construction of the two-dimensional atlas of gene expression patterns in situ.' In Silico Biology 2, Dagstuhl Seminar, 0011.
• A. Stathopoulos and M. Levine (2002). 'Dorsal gradient networks in the Drosophila embryo.' Developmental Biology 246, 57-67.
• A. Stathopoulos, M. Van Drenth, A. Erives, M. Markstein, and M. Levine (2002). 'Whole-genome analysis of dorsal-ventral patterning in the Drosophila embryo.' Cell 111, 687-701.
• P. Tomancak, A. Beaton, R. Weiszmann, E. Kwan, S. Shu, S.E. Lewis, S. Richards, M. Ashburner, V. Hartenstein, S.E. Celniker and G.M. Rubin (2002). 'Systematic determination of patterns of gene expression during Drosophila embryogenesis.' Genome Biology 3, issue 12: research0088.

General protocols:

• W. Sullivan, M. Ashburner, and R.S. Hawley, eds. (2000). 'Drosophila Protocols.' Cold Spring Harbor Laboratory Press.