, Incubate overnight at 4 C with HRP-coupled anti-DIG antibody (1/1000, Roche)

, Wash the embryos in KTBT (0.3% Tx) 10 times

, Wash the embryos for 1 min with the amplification buffer supplied by the manufacturer in the TSA kit

, Incubate the embryos with amplification buVer including freshly added FITC-labeled tyramide (1:100, Perkin Elmer) for up to 1 h at room temperature

, Wash in KTBT (0.3% Tx) twice for 5-10 min each at room temperature

, Detection of the Third RNA 1. Block the embryos by incubating with 15% sheep serum, 0.7% Roche blocking powder in KTBT (0.1% Tx) for 2-3, C. Protocol, vol.10

, Incubate overnight at 4 C with HRP-coupled anti-DNP antibody (1/500, Perkin Elmer)

, Wash the embryos in KTBT (0.3% Tx) 10 times

, Situ Hybridization Analysis of Chick Embryos in Whole-Mount and Tissue Sections 1. Dip the slides in 10% HCl/70% ethanol, followed by distilled water and 95% ethanol, for 1 min each

, Dip the slides in 2% TESPA (3-aminopropyltriethoxysilane) in acetone for 10 sec

, Wash twice with acetone, and then with distilled water

, Prehybridization Treatments Prior to hybridization, the sections are dewaxed, permeabilized by proteinase K treatment followed by refixation, and dehydrated. The probe is then spread over the sections under a coverslip, B. Protocol, vol.13

, Situ Hybridization Analysis of Chick Embryos in Whole-Mount and Tissue Sections 1. Place the slides in a slide rack and immerse in prewarmed 2 Â SSC, 0.1% CHAPS at 55-65 C until the coverslips fall oV, Gentle encouragement with forceps may be necessary

, Wash with 2 Â SSC, 0.1% CHAPS, twice for 30 min at 55-65 C

, Wash with 0.2 Â SSC, 0.1% CHAPS, twice for 30 min at 55-65 C

, Wash with KTBT, twice for 10 min at room temp

, Quickly drain each slide and place horizontally in a sandwich box containing moist tissue paper. Take care that the sections do not become dry, and quickly overlay them with 20% sheep serum in KTBT

R. Del-barrio, M. G. Nieto, and M. A. , Relative expression of Slug, RhoB, and HNK-1 in the cranial neural crest of the early chicken embryo, Dev. Dyn, vol.229, pp.136-139, 2004.

N. Denkers, P. Garcia-villalba, C. K. Rodesch, K. R. Nielson, and T. J. Mauch, Fishing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos, Dev. Dyn, vol.229, pp.651-657, 2004.

V. Hamburger, H. , and H. , A series of normal stages in the development of the chick embryo, J. Morphol, vol.88, pp.49-92, 1951.

C. Lopez-sanchez, V. Garcia-martinez, A. Lawson, S. C. Chapman, and G. C. Schoenwolf, Rapid triple-labeling method combining in situ hybridization and double immunocytochemistry, Dev. Dyn, vol.230, pp.309-315, 2004.

M. A. Nieto, K. Patel, and D. G. Wilkinson, In situ hybridization analysis of chick embryos in whole mount and in tissue sections, Methods Cell Biol, vol.51, pp.219-235, 1996.
URL : https://hal.archives-ouvertes.fr/hal-02912726

C. D. Stern, Detection of multiple gene products simultaneously by in situ hybridization and immunohistochemistry in whole mounts of avian embryos, Curr. Top. Dev. Biol, vol.36, pp.223-243, 1998.

D. G. Wilkinson, Whole mount in situ hybridization of vertebrate embryos, Situ Hybridization: A Practical Approach, pp.75-83, 1992.

D. G. Wilkinson and M. A. Nieto, Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts, Meth. Enzymol, vol.225, pp.361-373, 1993.

, Situ Hybridization Analysis of Chick Embryos in Whole-Mount and Tissue Sections