Abstract
Staining by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) typically detects activity of E. coli β-galactosidase (β-gal) in transduced tissues that express the LacZ reporter gene. In lung tissue from mice that received β-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 β-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.
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Acknowledgements
This work was supported by grants from the Cystic Fibrosis Foundation (CFF R881) and the NIH (NHLBI P01 HL59407, NHLBI PEGT U01 HL66948, NIDDK P30 DK47757). We thank the Vector and Quality Control Cores of the University of Pennsylvania for their help. JMW is an inventor on patents licensed to various commercial entities.
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Bell, P., Limberis, M., Gao, G. et al. An optimized protocol for detection of E. coli β-galactosidase in lung tissue following gene transfer. Histochem Cell Biol 124, 77–85 (2005). https://doi.org/10.1007/s00418-005-0793-2
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DOI: https://doi.org/10.1007/s00418-005-0793-2