On Mon, 10 Nov 1997, Arle Kruckeberg wrote:
> Hi Mike,
>> I've seen a few of your posts about yeast expression of GFP, and thought
> I'd put a technical problem to you.
>> We are expressing fusions of GFP with yeast plasma membrane
> proteins...
Dear Arle,
I want to stress that my recent post to the fluorpro group was a protocol
that is designed to achieve maximum GFP signal for reporter gene
expression determination by FACS in tissue culture cells. Fixation for
good preservation of subcellular localization of GFP fusions in cells will
likely take longer treatments with higher concentrations of fixatives.
> when we fix GFP-expressing cells and process them for immunofluorescence
> microscopy the GFP signal is significantly attenuated. We use more or
> less the conditions described in John Pringle's article in MetEnz vol
> 194, and have varied the time and temp of fixation...
That's the best approach, in general the best compromise between signal
loss and good preservation of ultrastructural detail must be empirically
determined. You are also running up against what almost everyone who has
ever looked at a GFP fusion in any living cell vs a fixed cell has
realized. Fixed material can often appear vastly different from live
cells!! Mark Flory and I never could find a suficiently mild fixation
technique for microtubules that would allow simultaneous visualization of
S. pombe GFP-CaM.
>...in your protocol the formalin exposure is so
> short, and that you incubate in PBS without formaldehyde before further
> processing (and you still presumably get good cross-linking)...
Probably not in that short of a fixation. (See above)
I would recommend a different protocol for yeast. The problem with yeast
is that it has the cell wall which can make fixation less than
instantaneous. Try using a higher concentration ~4% of paraformaldehyde
(make fresh from crystal) +/- 1/10th conc glutaraldehyde. If you can get
good fixation at 4 C sometimes it helps.
Aldehyde fixation is inherently unstable and with time subcellular
structures do tend to decompose so if you are interested in the best
figures possible, I would try to process samples as fast as possible prior
to imaging.
> Are prolonged exposures to formaldehyde deleterious?
Yes, for example if I fix my tissue culture cells then store them O/N in
fixative the fluorescence of my GFP positive cells will decrease.
> ...the Pringle mounting medium contains p-phenylenediamine to minimize
> photobleaching. This is, I think, a reducing agent. Could it be the
> culprit of our reduced EGFP signal? Or do you use it too?
Back when J.P. wrote that it was OK, but phenylene diamine sucks as a
photobleaching inhibitor. Try Citifluor-Glycerol, "mountant media #0",
from Ted Pella, Inc., P.O Box 492477, Redding, CA 96049 USA Cat #19470.
It really works well with GFP as well as conventional fluorochromes such
as FITC, Texas Red, etc. There are probably similar competing products
from other vendors, as well.
>> ps Hows the weather in my old home town?
When were you here and whom did you work with?
Meteorologists say we are supposed to be experiencing the effects of the
El Nino this year. The skies have been fairly clear with a dry winter so
far. Today clear and sunny temp ~20 C. We were skiing by this time last
year and the slopes aren't even close to being opened up in 1997.
Good luck with it.
MM
Mike Moser Tel: 206-543-6585
UW Department of Pathology FAX: 206-543-3967
Box 357705 moser at u.washington.edu
Seattle, WA 98195 http://weber.u.washington.edu/~moser
