Double fluorescent in situ hybridization to zebrafish embryos

• Published in: Trends in genetics Vol. 12; no. 10; pp. 387 - 389
• Main Authors: Jowett, Trevor, Yan, Yi-Lin
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01-10-1996; Elsevier Science
• Subjects: Animals; Biological and medical sciences; Danio rerio; Diverse techniques; Embryo, Nonmammalian - cytology; Embryo, Nonmammalian - physiology; Fluorescent Dyes; Freshwater; Fundamental and applied biological sciences. Psychology; In Situ Hybridization, Fluorescence - methods; Indicators and Reagents; Molecular and cellular biology; RNA Probes; Zebrafish - embryology; Embryonic development; Molecular hybridization; Vertebrata; Brachydanio rerio; Embryo; Gene; Pisces; In situ; Coexpression; Method; Detection; Gene expression
• ISSN: 0168-9525
• DOI: 10.1016/S0168-9525(96)90091-8

To understand the genetic networks that regulate development, it is often useful to know whether individual cells are simultaneously expressing more than one gene suspected to be involved in a particular process. This question can be solved by two-colour in situ hybridization using chromogenic substrates in tissue sections or whole-mount embryos. If the domains of expression overlap, however, the staining of one chromogen can mask that of the other. We have overcome this problem by using fluorescent substrates for alkaline phosphatase-conjugated antibodies. We have tested three different Fast Red substrates: (1) Vector Red alkaline phosphatase substrate (Vector Laboratories); (2) Fast Red tablets (Boehringer Mannheim); and (3) Sigma Fast Fast Red TR/Naphthol AS-MX (Sigma). Each substrate has been used in combination with the enzyme labelled fluorescence substrate (ELF, Molecular Probes). One probe is labelled with fluorescein, and the other with digoxigenin, and they are visualized by sequential incubation in alkaline phosphatase-conjugated antibodies to fluorescein and digoxigenin, respectively. The first antibody is stained with one of the three Fast Red formulations and the second with the ELF substrate. The signals are viewed by fluorescence microscopy with rhodamine and DAPI (4,6-diamino-2-phenylindole) filter sets, respectively. Molecular Probes supplies a variety of kits containing ELF substrate. We use the substrate formulation in the ELF-AP immunohistochemistry kit (E-6600).