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LacZ Transgenic Mice and Immunoelectron Microscopy: An Ultrastructural Method for Dual Localization of ß-Galactosidase and Horseradish Peroxidase

Patricia L. St. John and Dale R. Abrahamson

Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas

Correspondence to: Dale R. Abrahamson, PhD, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 3038, Kansas City, KS 66160. E-mail: dabrahamson{at}kumc.edu

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, ß-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of ß-galactosidase and peroxidase reaction products within mouse kidney. After incubation of transgenic LacZ tissues with IgG–HRP conjugates, samples were lightly fixed with 2% paraformaldehyde and 0.4% glutaraldehyde and processed for peroxidase histochemistry. Tissues underwent ß-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin ß1 chain IgG–HRP specifically to developing glomerular basement membranes, whereas Flk1/LacZ was expressed only by glomerular endothelial cells. In Epas1/LacZ mice, we immunolocalized anti-platelet endothelial cell adhesion molecule-1 specifically to glomerular endothelial plasma membranes, whereas Epas1/LacZ was expressed by both glomerular endothelial and mesangial cells. This dual ultrastructural localization technique should be broadly applicable for immunoelectron microscopic studies in LacZ transgenic animals, particularly those where LacZ expression and antibody–HRP binding are both relatively abundant. (J Histochem Cytochem 55:1207–1211, 2007)

Key Words: ß-galactosidase histochemistry • Epas1 • Flk1 • endothelial cells • glomerular basement membrane • horseradish peroxidase • laminin • mesangial cells • platelet endothelial cell adhesion molecule-1 • peroxidase immunohistochemistry

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