Embedding Embryos for High-Resolution Episcopic Microscopy (HREM)

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. For HREM, the choice of fixative appears to be of little consequence. If gene expression patterns are to be visualized in addition to tissue architecture, whole-mount staining for gene expression patterns using the 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) or LacZ detection system must be performed before embedding of samples. This protocol describes the procedure for embedding E11.5 mouse embryos for HREM imaging. Processing times should be adapted for the size and nature of other specimens.