Author/Editor		Jež, Mojca; Bas, Tuba; Veber, Matija; Košir, Andrej; Dominko, Tanja; Page, Raymond; Rožman, Primož
Title		The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem
Type		članek
Source		Histochem Cell Biol
Vol. and No.		Letnik 139, št. 1
Publication year		2013
Volume		str. 195-204
Language		eng
Abstract		Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. (Abs. trunc. at 2000 ch.)
Descriptors		TESTICULAR NEOPLASMS IMMUNOHISTOCHEMISTRY STAINING CHLORIDES FLUORESCENCE GLYCERIN MICROSCOPY, PHASE-CONTRAST TUMOR CELLS, CULTURED DYES DNA PHOTOBIOLOGY