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Dear Pauline,

Keeping your sample free from light is always a good idea and it is also
advisable to use some sort of "anti-fade" agent in your monting media.  We use
the following media:

20mM Tris pH 8.0
0.5% N-propyl gallate
90% Glycerol
Store at 4oC

This seems to work quite well.  Additionally, many types of mounting media
that are comercially available contain chemicals that prolong the life of your
fluorescent samples.  Good luck!

Mike


>
>pauline yu wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear List-peoples,
>> I'm quasi-self taught when it comes to using confocal, so please bear with
me for asking an elementary question. When it comes to working with samples
after they've been stained(in my case, Syto 24 for DNA and Alexa Fluor568
antibody), is it unsafe(i.e. damaging to the fluorochromes) to use a "darkroom
safelight" or a red light bulb to illuminate samples when trying to mount
them? I figured that if the wavelength of the red light was substantially
longer than the excitation wavelength, I wouldn't have to worry too much about
bleaching. Is this totally untrue? Should I just be working under the
*absolute minimum illumination* with my samples prior to imaging?
>>
>> My main problem is that I'm seeing very rapid bleaching of my Syto dye,
from just scanning a z-series through my sample(sea urchin eggs and embryos)
even though I'm using only 1-3% transmission(ancient Biorad MRC600). The only
other possibility I could think of is that my dye lot is "old"--purchased
about a year ago, but stored concentrated at -20C this whole time until before
use. My samples are mounted in 70% glycerol. Should I invest in some
fluorochrome stabilizing media or try a different mounting medium? My mounted
slides aren't nearly as "archival" for imaging purposes as I'd like them to
be.
>>
>> Many thanks for any pointers. Literature recommendations are greatly
appreciated.
>>
>> Pauline Yu
>> [log in to unmask]
>> Graduate Student
>> Manahan Research Laboratory
>
>--
>Dr. Andrea J. Elberger
>Professor, Anatomy and Neurobiology
>Director, Confocal Laser Scanning Microscope Facility
>The University of Tennessee, Memphis
>855 Monroe Avenue
>Memphis, TN  38163  U.S.A.
>tel: 901-448-4101
>FAX: 901-448-7193
><mailto:[log in to unmask]>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Michael C. Adams
Research Technician for Dr. Clare Waterman-Storer
Laboratory of Cell Motility Studies
Department of Cell Biology
The Scripps Research Institute
10550 North Torrey Pines Road, CB 163
La Jolla, CA 92037

TEL 858.784.9244
FAX 858.784.7521
EMAIL [log in to unmask]
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