Improved in situ beta-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of beta-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) with 5-bromo-indolyl-beta-o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial beta-galactosidase (lacZ). After beta-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for beta-galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of beta-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.