Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue.
The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine-potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.
Institute of Anatomy (CM, AMR), University of Leipzig, Leipzig, Germany.
Claudia
C
Merkwitz
Division of Urology, Department of Surgery, McGill University, Montreal, Québec, Canada (OB).
Orest
O
Blaschuk
Institute of Biochemistry (JW, AS, SP), University of Leipzig, Leipzig, Germany.
Jana
J
Winkler
Institute of Biochemistry (JW, AS, SP), University of Leipzig, Leipzig, Germany.|IFB AdiposityDiseases (AS), University of Leipzig, Leipzig, Germany.
Angela
A
Schulz
Institute of Biochemistry (JW, AS, SP), University of Leipzig, Leipzig, Germany.
Simone
S
Prömel
Institute of Anatomy (CM, AMR), University of Leipzig, Leipzig, Germany.
Albert Markus
AM
Ricken
D000818:Animals; D004396:Coloring Agents / Q000737:chemistry*; D004847:Epithelial Cells / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005697:Galactosides / Q000737:chemistry*; D017930:Genes, Reporter; D006651:Histocytochemistry / Q000379:methods; D007763:Lac Operon; D051379:Mice; D008822:Mice, Transgenic; D009928:Organ Specificity; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000737:chemistry*; D014468:Umbelliferones / Q000737:chemistry*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D004396:Coloring Agents; D029968:Escherichia coli Proteins; D005697:Galactosides; D013778:Tetrazolium Salts; D014468:Umbelliferones; C101993:cyclohexenoesculetin-beta-galactoside; D001616:beta-Galactosidase
LacZ expression; Salmon-Gal; epithelial appendages; filiform papillae; hair fibers; keratinized stratified squamous epithelia; penile spines; tetrazolium salts; β-galactosidase