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  • 标题:Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate
  • 本地全文:下载
  • 作者:Tadanobu Takahashi ; Tadamune Otsubo ; Kiyoshi Ikeda
  • 期刊名称:Biological and Pharmaceutical Bulletin
  • 印刷版ISSN:0918-6158
  • 电子版ISSN:1347-5215
  • 出版年度:2014
  • 卷号:37
  • 期号:10
  • 页码:1668-1673
  • DOI:10.1248/bpb.b14-00456
  • 出版社:The Pharmaceutical Society of Japan
  • 摘要:

    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-[3 H ]-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.

  • 关键词:phosphatase; fluorescent imaging; histochemical staining; benzothiazolylphenol; fluorescent substrate
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