Methods in Molecular Biology Volume 411, 2007, pp 13-23

Immunohistochemical Detection of β-Galactosidase or Green Fluorescent Protein on Tissue Sections

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With the recent advances in mouse genetics, it is now possible to mark specific cell types genetically in vivo and to study the fate of cells during development and adulthood. Cells are labeled and followed in vivo through the stable expression of reporter genes in particular cell types. The two most commonly used reporter genes are LacZ, which encodes the enzyme β-galactosidase (β-gal), and green fluorescent protein (GFP). β-Gal expression can be detected enzymatically, using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as a substrate, and GFP can be directly visualized by fluorescence microscopy. However, with single detection of β-gal or GFP, it is often impossible to determine whether expression of the reporter protein is restricted to a particular cell type. To ascertain the identity of individual cells within a multicellular tissue, β-gal or GFP proteins must be visualized in conjunction with additional cellular markers. For such experiments, specific antibodies raised against β-gal or GFP can be used in coimmunofluorescence analyses. Such double-staining analyses on tissue sections are a powerful tool to study transgene expression or to trace cells in multicellular tissues.