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Hi Yevgeniy,
Another possibility is that your mounting medium has aged to the point
where it promotes photo-oxidation (or reduction?) of DAPI to a green
fluorescent molecule(s). Another possibility is that you (or someone you
work with) did not dilute the DAPI as much as usual.
Enjoy,
George
p.s. your - and other's - copyright notices are pretty silly for
listserv postings.
-------- Original Message --------
Subject: Plastic Chamber Slides
Date: Mon, 25 Apr 2011 12:30:28 -0400
From: Yevgeniy Romin <[log in to unmask]>
Reply-To: Confocal Microscopy List <[log in to unmask]>
To: [log in to unmask]
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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?
---------------------------------------------------
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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