Nailpolish. was: Losing GFP Signal
Steffen Dietzel
wheras I never had problems with overnight fixation of cells in 1.6 %
formaldehyde, I did have problems with the nailpolish: I have tried several
ones and they all killed the GFP before the slide was even on the scope. I have
heard rumors of nailpolish that does not harm GFP, but was never able to hunt
one down, therefore PLEASE:
Could you let us know the exact brand name, product number or whatever is
usefull to find the nailpolish you are using?
Thanks! Steffen
--- Anda Cornea <corn...@OHSU.EDU> schrieb:
>
> >>> "Goodhouse, Joseph" Long
> For fixation employ low percentage paraformaldehyde for the shortest periods
> of fixation as possible( 5- 10 minutes for cells). Samples should be
> mounted in glycerol based mounting medium. I have found 90% buffered
> glycerol to be best. Avoid using nail polish as sealant of coverslips. In
> a short time this will wipe out your GFP signal.
>
> We routinely use 4%paraformaldehyde, 30minutes fixation, for cells expressing
> a GFP-membrane receptor chimera and seal slides with nailpolish. We see no
> loss of intensity when compared with live cells expressing the same
> construct, not even after a few months storage.
>
=====
Dr. Steffen Dietzel
Dept. of Cell and Structural Biology
University of Illinois at Urbana-Champaign
601 S. Goodwin Ave.
Urbana, IL 61801
Phone: +1/217/333-8372 Fax: +1/217/244-1648
e-mail: diet...@yahoo.de Web: http://www.life.uiuc.edu/belmont/dietzel
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Robert Palmer
Creme (hypo-allergenic - maybe that's the secret!). The shade is Canyon.
this deos not kill GFP in Streptococcus gordonii or Mycobacterium
smegmatis. I actually prefer Bloodorange however. It goes better with my
accesories...
Rob
Tamara Howard
Hasn't destroyed GFP to the best of my knowledge. Also works on regular
immunofluorescence slides. We mount in buffered glycerol with
paraphenylenediamine.
By the way - has anyone looked at confetti-type nail polish under
brightfield? Pretty nifty.
Tamara Howard
CSHL
Jacques Paysan
I found some melted parafin most useful to replace nail polish (I picked
this up from a newsgroup discussion some time ago). Simply apply with the
edge of a heated slide that you dip into a block of parafin. With a little
practice this works very easily. No effect on GFP and other fluorescent dyes
at all.
Jacques
Anda Cornea
I use clear Wet 'n Wild. It is toluene/formaldehyde free, I wonder whether this is what makes a difference.
Clonetech recommends rubber cement or agarose for sealing, but we found no reason to not use nailpolish, our standard sealant.
Anda Cornea, Ph.D.
Oregon Regional Primate Research Center
505 NW 185th Avenue
Beaverton, OR 97006
ph: (503) 690-5293
fax:(503) 690-5384
>>> Jacques Paysan <pay...@UNI-HOHENHEIM.DE> 03/10 2:42 PM >>>
Edward Monosov
fl.oz. Works great.
--
Edward Monosov, Ph.D.
Director
Cell Analysis Facility
The BURNHAM INSTITUTE
10901 N. Torrey Pines Rd, La Jolla, CA 92037
Tel: (858) 646-3100
Office: r. #5144, ext. x3206
Confocal Microscopy: r. #5105, ext. x3466)
Fluorescent Microscopy: r. #5121
Electron Microscopy: r. #5123),ext. x3620
FAX (858) 646-3196;
E-mail: emon...@burnham-inst.org
Louis M Kerr
The following is a sealant called VALAP and originated by Bob Allen in
60's. The VALAP recipe is: 1/3 paraffin (56 degree type works well), 1/3
vaseline, 1/3 lanolin by weight, lightly heat and mix. Carefully melt and
stir. IT IS FLAMMABLE. It is nice because it is easy to make, stores well
as a soft solid and is easy to apply to seal coverslips with a artist's
paint brush after lightly heating to a liquid. Be careful because it burns
easily! With the help of Nina Allen and Bob Hard I was able to locate at
least one of the original references. It is: McGee-Russel, S and Allen,
RD (1971). Reversible stabilization of labile microtubules in the
reticulopodial network of Allogromia. Advances in Cell and Molecular
Biology 1: 153-184. It mentions the VALAP recipe and the metal slide idea
used to hold double coverslip preps. The article also mentions apparent
lack of toxicity from the VALAP. Nina mentions that there is permeability
at least of oxygen and that is why her cell types do so well with the VALAP
over longer time periods. As Bob Hard points out: "Regarding the aluminum
slides (actually in Bob's lab and in mine, they were/are made from brass,
which doesn't bend as much)..." The article also describes the design of
an elegant microperfusion chamber and a technique for correlative light and
TEM. Reading the technique reinforces the need for an experienced lab hand!
Hopes this helps propel science down the road.
Louie
Louie Kerr
Research and Education Support Coordinator
Marine Biological Laboratory
7 MBL Street
Woods Hole, MA 02543
508-289-7273
508-540-6902 (FAX)
508-292-0289 (Cell phone)
VISIT OUR WEB SITE:
http://www.mbl.edu
Rosemary White
I would just like to emphasize the usefulness of Valap (re. message from
Louie Kerr), which I've been using to seal slides since student days. On
occasion, I've wanted a harder sealant or one that adhered better to wet
slides, and there are a number of dental waxes of varying hardness that
work very well for sealing in living tissue - like the dental wax used to
seal knife boats when sectioning. One we've used recently is Kemdent
sticky wax, made in the UK by Associated Dental Products - sets to hard
candle wax consistency. We've also used small drops of this dotted on the
corners of a coverslip to prevent squashing of fragile specimens, like
protoplasts. A soft sealant, consistency of plasticine, that also works
well is Surgident periphery wax from Heraeus Kulzer, Dental products
division, made in USA. Marvellous stuff!
no financial interest, etc, etc
cheers,
Rosemary
Rosemary White
Microscopy Centre
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
phone 61-2-6246 5475
fax 61-2-6246 5000
email r.w...@pi.csiro.au