12/5/97
>Molecular Probes ProLong, SlowFade, and SlowFade Light. I know it makes
>no logical sense, but in our experience fluorescent labels of rat tissue
>fade under the laser a whole lot faster than in human tissue.
>
CHRISTINE JONES
JONESCH@VAX.CS.HSCSYR.EDU
cytoskeleton within the cells of the embryo sac. I am using fixed tissue
embedded in a low melting point wax (Steedmans wax) which is removed with
alcohol prior to double labelling for tubulin (antibodies - FITC tag) and
DNA (Hoechst).
Anti-fade agents (glycerol- PDA) and Citifluor work quite well for FITC
but not at all for Hoechst, which fades and/or becomes non-specific
throughout the tissue within 24 hours. A 'no-antifade' solution which
is made up in glycerol (a mixture of biocarb buffers) and adjusted to pH
8.6 seems the best so far, but this is still not adequate to collect
images with uv after 3-4 days.
I'd be grateful for any help
Meredith Wallwork (Dr)
Department of Horticulture, Viticulture and Oenology
Waite Campus
University of Adelaide
Sth Aust
Meredith Wallwork
mwallwor@waite.adelaide.edu.au
does not quench shorter wavelength fluorophores such as DAPI and Hoechst.
Their Prolong Kit has also produced superior results in some applications
such as unfixed GFP expressing tissue. See their helpful handbook on line
at: http://www.probes.com/handbook/toc.html
go to Chapter 26.1
http://www.probes.com/handbook/ch26-1.html#ProLong
Larry D. Ackerman
Lily & Yuh Nung Jan Laboratories
Howard Hughes Medical Institute
UCSF, Box 0725, Rm U226
533 Parnassus Ave.
San Francisco, CA 94143
(415) 476-8751 FAX (415) 476-5774
mishot@itsa.ucsf.edu
Prolong Anti-Fade from Molecular Probes and love it. However, we did come
up with a very cheap media that has worked very well for double and triple
labels in cojuction with a DAPI stain.
70% glycerol
25% .5M tris pH 9.0
5% N-propyl gallate
Heat in a boiling water bath to dissolve the n-propyl gallate.
Cool and pH to 7.4
Keep light tight at 4 degrees C.
Use as little as neccessary to cover the coverslip.
Bob
Derm Imaging Center
U of W
Seattle
Robert Underwood
underwoo@u.washington.edu
>tests of various formulations (eg, DABCO, ppd, npropylgallate and others)
>and the winners were always prep dependent. There was no one of these that
>was a "sure thing". Also, there was an influence on which fluorophore was
>involved. I have no direct experience with testing commercial versions, but
>it certainly wouldn't surprise me if results with these too were
>preparation dependent.
> Just my two photons,
> Tobias Baskin
baskin@BIOSCI.MBP.MISSOURI.EDU
>a refractive index near Glycerol (I measured it with a
>refractometer), and seems to do a great job of preventing fading,
>although it works much better on rhoadmine compounds that fluorescein.
>
>Steve
Stephen T. Haley, II
SHALEY@DCSMSERVER.MED.SC.EDU
> In response to Christine's question about antifade reagents, I
extensively
>use Prolong Antifade, and get, in general, very good results on cell
>culture preps (my usual dyes are Oregon Green and propidium iodide or
>relatives). However, let me take advantage of the subject being on and ask:
>has anyone had occasional trouble with it? Because I have. Sometimes, with
>no apparent reason (read as: "I don't know, but someone may"), the mounted
>slides turn yellowish as seen directly by the eye, and under the
>microscope, everything is fuzzy, almost as if the fluoresent dyes had
>"leaked" and were evenly distributed throughout the prep in and out of the
>cells. It looks really bad, and as I said, I cannot identify the cause and
>in this way I lose preps at random. HELP!
>
Susana Zanello
szanello@BU.EDU
>glycerol, a recipe Steve Kempf gave me. Works great. Not good for living
>cells, though, obviously!
>>* Anthony Moss voice (334)844-9257 *
>* 101 Cary Hall fax (334)844-4065 *
>* Zoology and Wildlife Science email mossant@mail.auburn.edu *
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vs rat
>observation, but I was advised by someone at Leica that the freshness of the
>prep was really important. The fresher the prep the bigger the problem with
>bleaching. Maybe your rat tissues are taken and used sooner post mortem
than the
>human stuff? Anyway,one recommendation which was made to me to reduce bleaching
>was to leave the mounted specimens at least a day before viewing. I guess as
>with most things there will be a trade off against the deterioration of the
>specimen with time. I use mowiol so I have to leave the preps at least
overnight
>to set. From what I understand, bleaching is all a problem of oxygen and
>oxidative free radicals, therefore one of the things which I do is to
degass the
>mountant! I have never directly compared degassed vs normal, but I am in
general
>very happy with the stability of fluorescence in my coverslips. With mowiol
>there is of course the problem of the cells becoming a bit flattened by the
>compression of the mowiol when it dries, but as I do not do 3D reconstructions,
>this minor irritation is more than compensated for by the ease of use and
>stability.
>I hope this is some help
>Matthew
>
>--------------------------------------------------------
> Dr. Matthew J Hannah
> Institute for Neurobiology
> University of Heidelberg
> Im Neuenheimer Feld 364
> D-69120 Heidelberg
> Germany
>
> tel: +49 (6221) 54-8320 or 54-8692
> fax: +49 (6221) 54-8301
>
> http://server.nbio.uni-heidelberg.de/neurobiology.html
>suppressing initial intensity. SlowFade light is supposed to have remedied
>that, but I have not tried it. Personally, I prefer Permafluor from Lipshaw
>(Detroit) because it is water based and sets hard. If I don't need a
>permanent mount, I use a glycerol n-propyl-gallate mixture.
>
>David Carter
>InSight Biomedical
MiiCarter@AOL.COM
>antifade mountant comes from the following reference,
>
>Giloh,H. and J.W. Sedat. 1982. Fluorescence microscopy: reduced
>photobleaching of rhodamine and fluorescene protein conjugaes by n-propyl
>gallate. Science. Vol. 217: pp. 1252-1255.
>
>The recipes given are tailored to either FITC or RITC; however, I suspect
>that this formula will work well with other fluors as well. For instance,
>I've used it with Texas Red.
>
>Steve Kempf
kempfsc@MAIL.AUBURN.EDU
>We've never used commercial mountants but our recipe is:
>
>Anti fade mountant. Dissolve 0.1 - 0.01% p-phenylenediamine (PPD) (Sigma
>#P1519) in 50 - 100% glycerol in PBS. We are presently using 0.01% but
>this varies between batches. Aliquot and store frozen -80oC. If it goes
>dark throw it out. The glycerol % is dependent on the size of the whole
>mount you are coversliping, 50% for tiddlers, 100% for biggies - to stop
>too much squashing.
>Cheers,
>Eric Hines
>Microscopy Centre
>CSIRO Entomology
>Canberra Oz
Eric Hines
erich@ENTO.CSIRO.AU