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Hi Yevgeniy,
I have seen this effect with cells grown on glass coverslips as well, usually when the dye is present in very high concentration, so I'm not sure that it is related to the plastic slides. It has also been previously reported to this listserver.
The only solution I have found in this case is to minimise viewing of the DAPI by eye with the microscope (using excitation from the Hg or Xe arc lamp) prior to acquiring with the confocal system or camera and image the green fluorophore (Alexa488) first if possible.
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND
Tel: 64 9 373 7599 Ext 87438
Fax: 64 9 373 7484
http://www.fmhs.auckland.ac.nz/sms/biru/
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Yevgeniy Romin
Sent: Tuesday, 26 April 2011 4:30 a.m.
To: [log in to unmask]
Subject: Plastic Chamber Slides
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Hello List
We recently did an experiment where cells were stained with DAPI and Alexa 488. These cells were grown on plastic chamber slides, then fixed and stained. During imaging, we started seeing very weird bleaching patterns and bleedthrough of DAPI into the green viewing filter, even while just viewing the slides with a regular wide-field microscope. We used the same staining protocols as always (mounting media with anti-quenching reagents, etc.). The antibody that we used always worked well in our hands. The only difference that I can think of this time is the plastic slide. We haven't done staining on those before. Does anybody have any similar experience with staining cells on plastic chamber slides?
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Yevgeniy Romin
Digital Microscopist
Memorial Sloan-Kettering Cancer Center
Molecular Cytology Core Facility
1275 York Ave. Box 333
New York, NY 10065
Tel.646-888-2186
Fax. 646-422-0640
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