Clearing and photography of whole mount X-gal stained mouse embryos.

Advances in genetic manipulation in mice have made knockout, knock-in, and transgenic mice highly useful for elucidation of gene function and structure. In many cases the mice are engineered to include a reporter gene, for example, β-galactosidase (lacZ) or green fluorescent protein (GFP). The reporter gene enables visualization of the expressing cells and tissues in both the heterozygous and mutant animals. When looking for promoters and enhancers that direct gene expression in a spatiotemporal-restricted manner, the ability to easily detect expression derived by various genomic fragments is essential. Traditionally, 5-bromo-4-chloro-3-indolyl-β-d-galactopy-ranoside (X-gal) staining of mouse embryos younger then 16 days (E16) is performed as a whole mount. X-gal staining of developing organs of young embryos (e.g., less than E12) is easily viewed under a dissecting microscope as the thickness of the embryo does not obscure the staining. In older embryos (e.g., E16) stained cells close to the surface are easily detected, while deeper staining appears blurred, making sectioning and microscopic examination necessary. In addition to sectioning, organs of older embryos can be dissected, X-gal stained, and visualized on a dissecting scope (1). Here we describe two simple manipulations that increase the visibility of the stained tissues. During our attempts to improve photography of later-stage, alizarin red-and alcian blue-stained skeletons, we noticed that the quality of the images, the distinction between the colors, and the ability to observe the fine details are much improved when using dark field, in comparison to the conventional bright field illumination (Figure 1, A–D). Differential staining of bone and cartilage was performed according to a published protocol (2). In brief, E16.5 embryos were deskinned and eviscerated, stored in 100% ethanol, and transferred to acetone. After 48 h, they were stained overnight at 37°C in a staining solution consisting of 1 volume of alizarin red S (Sigma, St. Louis, MO, USA; 0.1% w/v in 95% ethanol), 1 volume of Alcian blue 8GX (Sigma; 0.3% w/v in 70% ethanol), 1 volume of concentrated acetic acid, and 17 volumes of 70% ethanol. The specimens were briefly rinsed in water and cleared in 1% w/v KOH, followed by 20% glycerol solution (1 volume glycerol and 4 volumes of 1% KOH) and by graded steps of glycerol/1% KOH solutions with increasing amounts of glycerol (50%, 80%, and 100%) by incubating for a week in each solution at room temperature. As expected, an E16.5 embryo became translucent, and the developing bone (red) and …