Mounting media -- PVA + DABCO?
Kathy Terry
New, quick question from the dumb blonde: Does anyone mount their
specimens in a polyvinyl alcohol mixture that includes DABCO? The folks
around here use PVA with p-phenylenediamine in phosphate buffered saline.
This mount hardens after a few hours, but I am concerned with distortion
effects attributed to PVA. Can anyone supply a recipe that produces a
mount which will harden (I'm making whole mounts of insect tissues) and
includes DABCO? Thank you kindly!
Kathy
Katherine L. Terry
York University
Biology Department
4700 Keele St
Toronto, Ontario
M3J 1P3
(416)736-2100 ext 20649
Johan Wassélius
Dear Kathy and fellow confocalists!
Yes, I mount my specimens using PVA-DABCO, and I am very happy doing so.
The medium polymerizes upon contact with air forming a strong seal at the
perimeter of the coverslip. If you absolutely have to get the coverslip off
you can do so by soaking the slide in buffer.
I have not noticed that it, compared to other mounting medias, should cause
any distortion (though I have not spent years and years studying the matter).
Anyway, this is our handy-dandy guide to 50 ml of PVA-DABCO, we originally
got it from Dr. Daniel Peterson at Chicago Medical School.
Day 1
1. Add 4.8 g of polyvinyl alcohol (PVA) to 12 g of glycerol and mix well.
2. Add 12 ml of distilled water and leave it on a rotator at room
temperature over night.
Day 2
3. Add 24 ml of 0.2M Tris-HCL at pH 8-8,5.
4. Heat in a water bath to 50°C while mixing for about 30 minutes.
5. Add 1.25 g of DABCO and mix well.
6. Centrifuge at about 2000 rpm for 5 minutes.
7. Aliquot the supernatant (we use 1 ml aliquots which is enough for 15-20
slides) and store at -20°C.
Since it polymerizes upon contact with air you should not refreeze the
aliquots. Thaw it just before use and throw away what you do not use. Sigma
has both the PVA (#P8136) and DABCO (#D2522), and so has Chemicon (#151938
RT for PVA and #195137 for DABCO).
I hope this is useful for you, Kathy, and others. Please do not hesitate to
contact me if I have missed any information.
Cheers,
Johan
~~~~~~~~~~~~~~~~~~~~
Johan Wassélius
Wallenberg Retina Center
Department of Ophthalmology
Lund University Hospital
S-221 85 Lund, Sweden
Phone: +46-46-17 24 84
Home phone: +46-46-211 99 93
Cell phone: +46-0703-750593
Fax: +46-46-17 27 21
e-mail: johan.w...@oft.lu.se
~~~~~~~~~~~~~~~~~~~~
MiiC...@aol.com
say, pure DPX, Vectashield, Permafluor, or whatever you were using before? I
have an application where I image a very large area of slide quantitatively,
and have found that any antifade agent introduces a tiny amount of background
which can show itself over wide areas. The application is visible-only, but
covers the whole range from 488 to 633 Ex, 512-700 Em.
Do you know if this recipe has any resemblance to commercial preps like
ProLong?
Many thanks,
David Carter
Genomic Solutions (Lansing Division)
Johan Wassélius
I have not used a wole lot of other mounting medias. What I have used a lot
is Vectashield and another "home-brew" based on glycerin with
1,4-phenylenediamine (which is toxic). With the latter I see exactly what
you mention i.e. a background caused by the antifading agent (especially if
it takes a couple of days between the mounting and the examination of the
slide).
With Vectashield or PVA-DABCO I have subjectively not noticed any
particular background introduced by the mounting medium, compared to simply
mounting the slide in buffer and examine it right away. However, it does
not mean that there is no background whatsoever. If your application is
more sensitive then mine, you might find problems in situations where I am
totally happy, I hope that is not the case here.
As for other mounting medias such as ProLong and others, I am sorry that I
cannot be of much help for you.
To state the obvious, you might have even more trouble not using
antifading, since the fluorophor might bleach very fast and that may
introduce an opposite error. And to continue the stating of obvious facts,
background can be introduced by a lot of other factors, such as fixation,
tissue preparation and so on. But since the background is visible in
several filters I think it should be possible to subtract the "backgroud
pixels" in your, say 568 channel, from your "speciffic and background
pixels" in your, say 488 channel, in specimens with unevenly distributed
background and end up with a specific image that might not be beautiful,
but might be good for quantitative analyzis. This is really not much more
then an idea I have, I have not tested it very much in real life so far,
but I think it may prove usefull in some applications, I am sure someone
has done it before.
Cheers,
Johan
>How much residual fluorescence do you see with this preparation, compared
to,
>say, pure DPX, Vectashield, Permafluor, or whatever you were using before?
I
>have an application where I image a very large area of slide quantitatively,
>and have found that any antifade agent introduces a tiny amount of
background
>which can show itself over wide areas. The application is visible-only, but
>covers the whole range from 488 to 633 Ex, 512-700 Em.
>
>Do you know if this recipe has any resemblance to commercial preps like
>ProLong?
>
>Many thanks,
>
>David Carter
>Genomic Solutions (Lansing Division)
~~~~~~~~~~~~~~~~~~~~
Tamara Howard
you will see fading and background. We buffer our medium with
bicarbonate to keep the pH up.
The glycerol you use makes a difference, too. Some glycerols will
autofluoresce...I have *no* idea why, but we found a reliable source
(after testing glycerol from several vendors) but always check a new
bottle before using to make mounting media.
My own observation as far as Vectashield goes - it is fine if you
are looking at freshly prepared samples, but we start to see a lot of
media autofluorescence once the slides have been stored at -20C. Slides
mounted with the para-phenylenediamine medium can be stored at -70C for
years and still be gorgeous on the 'scope. Prolong stores better than
Vectashield (in my experience) - but I'm convinced that it is
para-phenylenediamine-based, just from the way the dry ingredient looks
and behaves.
My $0.02
Tamara Howard
CSHL