Fixation/permeabilization procedure for mRNA in situ hybridization of zebrafish whole-mount oocytes, embryos, and larvae

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.