Dj. Weiss et al., Histochemical discrimination of endogenous mammalian beta-galactosidase activity from that resulting from lac-Z gene expression, HISTOCHEM J, 31(4), 1999, pp. 231-236
Minces of several organs from the transgenic mouse ROSA beta-gal 26 (ROSA-2
6), 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 rang
ing from 7.5 to 9.5 to determine the optimal pH for in situ demonstration o
f bacterial beta-galactosidase activity (neutral pH optimum) while minimizi
ng detection of potentially confounding endogenous mammalian beta-galactosi
dase (acidic pH optimum). Similar studies were performed with organ minces
from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and maca
ques to confirm the effect of pH on minimizing detection of endogenous mamm
alian beta-galactosidase. In all organs evaluated; heart, liver, spleen, ki
dney, brain, and skeletal muscle, endogenous beta-galactosidase activity wa
s rarely detected following incubation at pH greater than 7.5. In contrast,
bacterial beta-galactosidase activity in the ROSA-26 mice was strongly det
ected in organ minces following incubation at pH 8.0-9.0. These findings ar
e similar to previous observations we have made in lung minces and confirm
that a simple alteration of a commonly used histochemical technique for det
ecting in situ beta-galactosidase activity, raising the reaction buffer pH
to weakly alkaline range, can reliably distinguish between endogenous activ
ity and that resulting from exogenous bacterial gene expression.