3D Imaging of Gene Expression Domains in the Mouse Heart

In situ hybridization (ISH) is a powerful method for analyzing gene expression patterns in tissue sections and whole-mount specimens. Three-dimensional (3D) imaging of ISH signals helps to understand the gene expression patterns in the complex structure of developing organs. This chapter describes the protocol for creating 3D images of ISH signals in a cost-effective and time-efficient manner using the correlative microscopy and block-face imaging (CoMBI) system. A unique feature of the CoMBI system is the ability to obtain both serial block-face images and sections as needed for preferential staining, such as hematoxylin and eosin staining. Correlating section images and 3D data helps to annotate gene expression patterns and understand the anatomy of developing organs. We describe the protocol for the sample preparation (ISH and embedding), acquisition of serial block-face images, and image processing to create 3D images.