Solution => DRAQ5 or DRAQfx FIX & GO
Why? NO photo-switching, NO spectral overlap, NO freeze-thaw storage, and PROVEN in thousands of peer-reviewed papers.
Try the introductory pack size (DR50050) today and find out why DRAQ5 has become the counterstain of choice for so many labs around the world, offering the choice of live-end point or fixed sample staining.
If you work only with fixed cells and tissue sections then the choice should be DRAQfx FIX & GO - the far-red counterstain in a ready-to-use benchtop dropper bottle!
WHERE TO BUY
See these recent papers describing the problem:
UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated form.
Żurek-Biesiada, et al. Cytometry Part A (2013) 83A: 441-451
The authors' closing remark is: "A less welcome aspect of the photoconversion of Hoechst 33258 and DAPI is the issue of generation of unexpected green fluorescence signals in microscopy specimens labeled with the UV-excited and some other green, yellow or red emitting dyes. Even a small dose of UV may cause an increase in the green fluorescence signal derived from the investigated DNA dyes. This emission can be mistaken for a green fluorescence emitted by a different, blue-excited probe like fluorescein, Alexa 488 or GFP."
The hazards of DAPI photoconversion: effects of dye, mounting media, and fixative, and how to minimize the problem.
Jež et al. Histochem and Cell Biology (2013) 139: 195-204
The authors conclude: "Bleed-through of fluorescence emission complicates the interpretation of the acquired image and can lead to false results particularly in cases of co-localization studies and fluorescence quantification. Use of alternative DNA dyes, such as DRAQ5 (Martin et al. 2005) can alleviate the problem. DRAQ5 is a far-red fluorescent DNA dye (excitation/emission 647/670 [actually 647/697]). It is a good choice for nuclear staining of fixed and live cells. DRAQ5 is useful for most bench-top, wide-field and confocal systems and due to its far-red emission it is spectrally ideally compatible with FITC-based fluorochromes."
Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation.
Karg & Golic. Chromosoma. 2017 Dec 12. doi: 10.1007/s00412-017-0654-5
The authors summarise: "These results, along with previous findings by others, indicate that appropriate care should be taken when performing experiments with multiple fluorochromes that include DAPI or a Hoechst dye as a nuclear stain. Green or red fluorescence that mimics the pattern of DAPI and Hoechst fluorescence should be considered suspect without appropriate controls, or verification using other DNA dyes.."
See these recent papers describing the problem:
UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated form.
Żurek-Biesiada, et al. Cytometry Part A (2013) 83A: 441-451
The authors' closing remark is: "A less welcome aspect of the photoconversion of Hoechst 33258 and DAPI is the issue of generation of unexpected green fluorescence signals in microscopy specimens labeled with the UV-excited and some other green, yellow or red emitting dyes. Even a small dose of UV may cause an increase in the green fluorescence signal derived from the investigated DNA dyes. This emission can be mistaken for a green fluorescence emitted by a different, blue-excited probe like fluorescein, Alexa 488 or GFP."
The hazards of DAPI photoconversion: effects of dye, mounting media, and fixative, and how to minimize the problem.
Jež et al. Histochem and Cell Biology (2013) 139: 195-204
The authors conclude: "Bleed-through of fluorescence emission complicates the interpretation of the acquired image and can lead to false results particularly in cases of co-localization studies and fluorescence quantification. Use of alternative DNA dyes, such as DRAQ5 (Martin et al. 2005) can alleviate the problem. DRAQ5 is a far-red fluorescent DNA dye (excitation/emission 647/670 [actually 647/697]). It is a good choice for nuclear staining of fixed and live cells. DRAQ5 is useful for most bench-top, wide-field and confocal systems and due to its far-red emission it is spectrally ideally compatible with FITC-based fluorochromes."
Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation.
Karg & Golic. Chromosoma. 2017 Dec 12. doi: 10.1007/s00412-017-0654-5
The authors summarise: "These results, along with previous findings by others, indicate that appropriate care should be taken when performing experiments with multiple fluorochromes that include DAPI or a Hoechst dye as a nuclear stain. Green or red fluorescence that mimics the pattern of DAPI and Hoechst fluorescence should be considered suspect without appropriate controls, or verification using other DNA dyes.."
that's tru, Hoechst and Dapi photoconverts like hell and become green so overlap with GFP..
ReplyDelete