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.