In situ histochemical detection of beta-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous beta-galactosidase activity.
Bacterial lacZ is one of the most commonly used reporter genes for assessing gene transfer to lung. However, lung contains endogenous beta-galactosidase (beta-Gal), which can confound estimation of exogenous lacZ expression by histochemical techniques (i.e., X-Gal) for in situ demonstration of enzyme activity. We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure as well as lung tissue processing and fixation techniques, and found that none of these could be used to distinguish between endogenous and exogenous beta-Gal activities. The mammalian and bacterial beta-Gal enzymes, however, have pH optima in the acidic and neutral ranges, respectively. Exposing whole lung, lung minces, or mounted frozen sections of lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detection of endogenous activity in lungs from a variety of species while preserving that resulting from bacterial enzyme activity in a transgenic mouse expressing lacZ. This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture. An appropriate buffer that maintains the desired pH throughout the duration of X-Gal exposure must be used.
Weiss
D J
DJ
Division of Pulmonary and Critical Care Medicine, Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, 98104, USA.
Liggitt
D
D
Clark
J G
JG
D000818:Animals; D001419:Bacteria / Q000201:enzymology; D015870:Gene Expression; D017930:Genes, Reporter; D006651:Histocytochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007763:Lac Operon; D008168:Lung / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D010215:Papio; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001616:beta-Galactosidase