Plastic Chamber Slides

[Romin, Yevgeniy/Sloan Kettering Institute]

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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.[phone]
Fax. [phone]
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[Jacqueline Ross]

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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: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin
Sent: Tuesday, 26 April 2011 4:30 a.m.
To: [hidden email]
Subject: Plastic Chamber Slides

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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.[phone]
Fax. [phone]
---------------------------------------------------


 
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     Please note that this e-mail and any files transmitted with it may be
     privileged, confidential, and protected from disclosure under
     applicable law. If the reader of this message is not the intended
     recipient, or an employee or agent responsible for delivering this
     message to the intended recipient, you are hereby notified that any
     reading, dissemination, distribution, copying, or other use of this
     communication or any of its attachments is strictly prohibited.  If
     you have received this communication in error, please notify the
     sender immediately by replying to this message and deleting this
     message, any attachments, and all copies and backups from your
     computer.