Cellular Resolution Expression Profiling Using Confocal Detection of NBT/BCIP Precipitate by Reflection Microscopy

The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.