Histochemical discrimination of endogenous mammalian beta-galactosidase activity from that resulting from lac-Z gene expression.
Minces of several organs from the transgenic mouse ROSAbeta-gal 26 (ROSA-26), which robustly expresses bacterial lac-Z in most tissues, were exposed to 4-bromo-5-chloro-3-indoyl-beta-D-galactopyrosanide (X-gal) at pH ranging from 7.5 to 9.5 to determine the optimal pH for in situ demonstration of bacterial beta-galactosidase activity (neutral pH optimum) while minimizing detection of potentially confounding endogenous mammalian beta-galactosidase (acidic pH optimum). Similar studies were performed with organ minces from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and macaques to confirm the effect of pH on minimizing detection of endogenous mammalian beta-galactosidase. In all organs evaluated; heart, liver, spleen, kidney, brain, and skeletal muscle, endogenous beta-galactosidase activity was rarely detected following incubation at pH greater than 7.5. In contrast, bacterial beta-galactosidase activity in the ROSA-26 mice was strongly detected in organ minces following incubation at pH 8.0-9.0. These findings are similar to previous observations we have made in lung minces and confirm that a simple alteration of a commonly used histochemical technique for detecting in situ beta-galactosidase activity, raising the reaction buffer pH to weakly alkaline range, can reliably distinguish between endogenous activity and that resulting from exogenous bacterial gene expression.
Weiss
D J
DJ
Division of Pulmonary and Critical Care Medicine, Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, USA.
Liggitt
D
D
Clark
J G
JG
D000818:Animals; D001921:Brain / Q000201:enzymology; D005697:Galactosides / Q000378:metabolism; D015870:Gene Expression; D006651:Histocytochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007763:Lac Operon; D008251:Macaca; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013154:Spleen / Q000201:enzymology; D001616:beta-Galactosidase / Q000032:analysis*
D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside