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Fixation/Permeabilization Procedure for mRNA In Situ Hybridization of Zebrafish Whole-Mount Oocytes, Embryos, and Larvae

  • Ricardo Fuentes
  • Juan FernándezEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)

Abstract

A new procedure for improved in situ hybridization of zebrafish whole-mount oocytes, embryos, and early larvae is described. The procedure relies on the simultaneous fixation/permeabilization of samples using formaldehyde as fixative and short C-chain aliphatic carboxylic acids, particularly glacial acetic acid, as permeabilizers. As compared with in situ hybridization performed with routine methods, our procedure is simpler and provides better structural preservation of cells and tissues, equivalent mRNA signals, and similar results in embryos of different developmental stages. It is hypothesized that during aldehyde fixation short C-chain aliphatic carboxylic acids modulate the rate of formation and/or destruction of methylene bridges established between cell proteins.

Key words

Fixation/permeabilization Formaldehyde/carboxylic acids Formaldehyde/acetic acid Whole-mount in situ hybridization Zebrafish in situ hybridization Carboxylic acids as cell permeabilizers 

Notes

Acknowledgments

We thank Drs. M.C. Mullins, F. Pelegri, V. Gallardo, A. Reyes, and L. Valdivia for donation of cDNA-inserted plasmids or riboprobes and V. Guzman for technical assistance. Dr. M.C. Mullins kindly provided her laboratory facilities to perform some experiments. Financed by the University of Chile.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cell and Developmental BiologyUniversity of Pennsylvania, Perelman School of MedicinePhiladelphiaUSA
  2. 2.Department of Biology, Faculty of SciencesUniversity of ChileSantiagoChile

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