Galactosidase-catalyzed fluorescence amplification method (GAFAM): sensitive fluorescent immunohistochemistry using novel fluorogenic β-galactosidase substrates and its application in multiplex immunostaining.

Multiplex immunohistochemistry/multiplex immunofluorescence (mIHC/mIF) enables the simultaneous detection of multiple markers in a single tissue section by visualizing the markers in different colors. Currently, tyramide signal amplification (TSA) is the most commonly used method because it is heat resistant to multiplexing. SPiDER-βGal (6'-(diethylamino)-4'-(fluoromethyl)spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3'-yl β-D-galactopyranoside), a novel fluorogenic substrate of β-galactosidase (β-gal) was reported recently. Its properties are favorable for application in sensitive mIF based on quinone methide chemistry. Combining SPiDER-βGal with its related substrates, a novel, sensitive fluorescent IHC method for formalin-fixed paraffin-embedded (FFPE) sections was developed, named the galactosidase-catalyzed fluorescence amplification method (GAFAM). Evaluation of GAFAM indicated the following characteristics: (1) the entire GAFAM procedure was complete within a few hours; (2) the optimal working concentration of the substrates was 20 μM; (3) the fluorescent product was heat resistant; (4) the GAFAM exhibited sensitivity comparable with that of TSA, which was higher than that of conventional IF; and (5) the GAFAM was applicable to mIF and multispectral imaging. GAFAM is expected to be applicable to IF (or mIF in combination with TSA), and is a promising tool for facilitating morphological research in various fields of life science.