Histochemical imaging of alkaline phosphatase using a novel fluorescent substrate

Histochemical visualization of phosphatase is exclusively required for Western immunoblotting and antigen-positive cell staining using an alkaline phosphatase (AP)-labeled secondary antibody. This detection has been performed by several reagents including 5-bromo-4-chloro-3-indolyl-phosphate (X-Phos), nitro blue tetrazolium (NBT), 3-(2'-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone (ELF® 97 Phosphate). We previously reported that 2-(benzothiazol-2-yl)-4-bromophenol bonded with N-acetylneuraminic acid (BTP3-Neu5Ac), enabled fluorescent histochemical visualization of sialidase activity. 2-(Benzothiazol-2-yl)-4-bromophenol (BTP3), which is formed from BTP3-Neu5Ac by sialidase reaction, is a crystalline, insoluble and stable fluorogenic compound, deposited at the site of enzyme activity. We developed a BTP3 phosphate ester (BTP3-Phos) for the purpose of fluorescent histochemical visualization of phosphatase activity. BTP3-Phos emitted fluorescence in a manner dependent on the concentration of the AP-labeled antibody. BTP3-Phos also enabled fluorescent histochemical visualization of AP-blotted dots in a manner dependent on the concentration of the AP-labeled antibody. The detection sensitivity of BTP3-Phos was estimated to be greater than that of the conventional method using X-Phos and NBT. Influenza A virus-infected cells were fixed and reacted with anti-influenza A virus antibodies and incubated continuously with an AP-labeled secondary antibody. BTP3-Phos stained the infected cells with distinct green fluorescence. These results indicate that BTP3-Phos can enable fluorescent immunohistochemical staining analysis using an AP-labeled antibody. BTP3-Phos would be beneficial for histochemical staining of AP activity, and may be applicable for multi-color staining or a cell sorter.