LacZ transgenic mice and immunoelectron microscopy: an ultrastructural method for dual localization of beta-galactosidase and horseradish peroxidase.
Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, beta-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 beta-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 beta-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin beta1 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.
St John
Patricia L
PL
Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 3038, Kansas City, KS 66160, USA.
Abrahamson
Dale R
DR
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D017930:Genes, Reporter; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000254:growth & development / Q000648:ultrastructure; D007763:Lac Operon; D007797:Laminin / Q000276:immunology; D051379:Mice; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1