Vector alkaline phophatase substrate Blue III: one substrate for brightfield histochemistry and high-resolution fluorescence imaging by confocal laser scanning microscopy.
A number of histochemical chromogenic substrates for alkaline phosphatase are commercially available and give reaction products with a range of colours for brightfield examination. Some of these reaction products are also fluorescent, exhibiting a wide excitation range and a broad emission peak. We report here that one of these substrates, Vector Blue III, yields a stable, strongly fluorescent reaction product with an excitation peak around 500 nm and a large Stokes shift to an emission peak at 680 nm. The reaction product can be excited using a mercury lamp with a fluorescein excitation filter or an argon ion laser at 488 nm or 568 nm, and the emission detected using a long-pass filter designed for Cy-5. Thus, a single substrate is suitable for brightfield imaging of tissue sections and high-resolution analysis of subcellular detail, using a confocal laser scanning microscope, in the same specimen.
Hollinshead
M
M
Sir William Dunn School of Pathology, Oxford, UK.
Sanderson
J
J
Vaux
D J
DJ
D000324:Adrenocorticotropic Hormone / Q000032:analysis; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D007124:Immunoenzyme Techniques; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000648:ultrastructure; D010902:Pituitary Gland / Q000737:chemistry; D051381:Rats; D013050:Spectrometry, Fluorescence
D000324:Adrenocorticotropic Hormone; D000469:Alkaline Phosphatase