The lacZ gene is frequently fused to heterologous promoters to reveal the expression of endogenous genes in transgenic mice (Bonnerot and Nicolas, 1993; Goring et al., 1987) and in the detection of genetic recombination events in cell fate mapping

INTRODUCTION The lacZ gene is frequently fused to heterologous promoters to reveal the expression of endogenous genes in transgenic mice (Bonnerot and Nicolas, 1993; Goring et al., 1987) and in the detection of genetic recombination events in cell fate mapping experiments (Joyner and Zervas, 2006; Petit et al., 2005; Watson et al., 2008). The most popular substrate for the detection of galactosidase is X-gal (5-bromo-4-chloro-3-indolyl--Dgalactopyranoside), a glycoside that is used in combination with potassium ferriand ferro-cyanide (FeCN). The -galactosidase assay occurs in two steps. In the initial step, -galactosidase induces the release of a soluble, colorless indolyl group from Xgal. Subsequently, two indolyl moieties form a dimer that is oxidized to form an insoluble blue indigo precipitate (Cotson and Holt, 1958; Pearson et al., 1963). The dimerization and oxidation steps are facilitated by ferric and ferrous ions, which serve as electron acceptors (Lojda, 1970). There are multiple chromogenic substrates for -galactosidase that can substitute for X-gal, these include Salmon-gal (S-gal) (6chloro-3-indolyl--D-galactopyranoside), Magenta-gal (5-bromo6-chloro-3-indolyl--D-galactopyranoside) and Bluo-gal (5-bromo3-indolyl--D-galactopyranoside) (Aguzzi and Theuring, 1994; Brunet et al., 1998; Kishigami et al., 2006; Pearson et al., 1963), as well as fluorescent substrates (Zhang et al., 1991). S-gal has been shown to be more sensitive than X-gal in early mouse embryos when used in combination with ferric and ferrous ions (Kishigami et al., 2006). The tetrazolium salts NBT (nitroblue tetrazolium), TNBT (tetranitroblue tetrazolium) and INT (iodonitrotetrazolium) are substitutes for potassium ferriand ferro-cyanide and precipitate, when reduced, to form colored formazan compounds (Altman, 1976). X-gal, in combination with NBT, produces a purple precipitate, combined with INT yields a dark red brick color and mixed with TNBT, an intense dark-brown product (Altman, 1976). This later combination was found to be more sensitive than the classic X-gal/FeCN indigogenic reaction in tissue sections (Gugliotta et al., 1992). In experiments using the traditional X-gal/FeCN assay, we noticed that the expression pattern of some lacZ-based transgenes did not mimic the pattern of expression of endogenous genes. One explanation for these results is that the -galactosidase assay is not sensitive enough to reveal the full range of expression of the transgene. Because of the potential implications of this possibility in the interpretation of lacZ-based transgenic experiments, we embarked on a search for a more-sensitive -galactosidase assay. To achieve this goal, we assayed S-gal in combination with the tetrazolium salts NBT, TNBT and INT as alternative substrates for -galactosidase detection and compared them with the classic Xgal/FeCN assay. Here, we report that S-gal in combination with TNBT is the most-sensitive combination and produces faster results than all the different substrate combinations tested for galactosidase detection. These results provide a novel alternative for -galactosidase detection that will facilitate the analysis of reporter lines or cell fate studies and avoid false-negative results.