An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer.
Staining by 5-bromo-4-chloro-3-indolyl-beta-D: -galactopyranoside (X-gal) typically detects activity of E. coli beta-galactosidase (beta-gal) in transduced tissues that express the LacZ reporter gene. In lung tissue from mice that received beta-galactosidase-expressing adeno-associated virus (AAV) vectors via intranasal inhalation, we observed only a low frequency of positive cells after X-gal staining in contrast to other reporter genes, such as alkaline phosphatase or green fluorescent protein. In this study, we systematically tested a number of parameters to improve the sensitivity of X-gal staining in lungs transduced with beta-galactosidase-expressing AAV2/5 vectors. We observed that the use of nuclear-targeted LacZ instead of cytoplasmic LacZ as the reporter gene substantially increases the number of positive cells after X-gal staining. The pH of the staining solution determines staining sensitivity and background staining with pH 7.0 resulting in high sensitivity and no background levels. Glutaraldehyde at 0.2% or 0.5% in PBS as fixative provides optimal results for X-gal staining. The alternative substrate, Bluo-gal, showed no improvement compared with X-gal but instead caused nonspecific background staining. We further stained intact fixed lungs with X-gal and processed them for paraffin embedding or cryosectioning, resulting in equal staining intensities. However, en bloc staining of intact tissues resulted in the absence of positive cells within deeper-located lung areas.
Bell
Peter
P
Gene Therapy Program, Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Limberis
Maria
M
Gao
Guangping
G
Wu
Di
D
Bove
Mark S
MS
Sanmiguel
Julio C
JC
Wilson
James M
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D003593:Cytoplasm; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000032:analysis / Q000378:metabolism; D006651:Histocytochemistry; D007763:Lac Operon; D008168:Lung; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling; D014162:Transfection; D001616:beta-Galactosidase / Q000737:chemistry*
D001616:beta-Galactosidase