Kathy-

I can only help with the last part of your question. For many years, I used
p-phenylenediamine dihydrochloride, but found that it darkened
significantly over short periods of time. (I stored it at -20 to -85
degrees to retard the darkening process). Then I tried Vectashield, and was
very disappointed with it, because it autofluoresced significantly. For the
last six months, I have been using DABCO, and it is far better than either
of the other two for my purposes. (I usually use DAPI or propidium iodide
for nuclei or chromosomes, and have FITC, rhodamine, and Cy5 signals in
various combinations). I store the DABCO at -20 degrees in small aliquots.
It is quite viscous, so I usually warm it to 37 degrees before pipetting.
Give it a try; I think you'll be pleased.

Karen

>I'm a grad student starting project involoving confocal study of
>neuropeptides in the brain of the insect Rhodnius prolixus.  I'm trying to
>decide what type of fixative to use on the whole mounts I plan to prepare.
>Most literature suggests using formaldehyde (2-3%) in a buffer.  Some
>epifluorescence papers I've read use 'a modified Bouin-Hollande solution'
>including 0.7% mercuric chloride and lacking acetic acid.  I've never seen
>this solution used for confocal studies.  Is there a reason for this?
>Also, I've talked to a few people who have suggested using 0.1M glycine to
>quench autofluorescence of formaldehyde after fixing.  Does anyone have a
>reference which might explain the virtues of this?  Is it even necessary?
>One last thing (I promise!) -- I've also been digging through papers
>looking for a mounting media which will reduce photobleaching.  Things
>I've come across include DABCO, and something called Fluoroguard from
>Biorad.  Can anyone offer information about the performance of these
>antifades?
>
>                                        Thanks!
>
>                                                Kathy T.


Karen M. Gustashaw. M.S.
Department of Genetics
Case Western Reserve University
Biomedical Research Building, Room 709a
2109 Adelbert Road
Cleveland, OH  44106

Telephone: (216) 368-3327
FAX: (216) 368-3432
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