[Histonet] Re: Long discussion on Immunofluorescence/slide storage
We cover the autostainer mindful the slides should not be exposed to light. We coverslip from water because of the salts in the buffers. The refrigerator light is burnt out and I refuse to replace it. (because of IF slides) It isn't trash on the slides exactly, it a light very light cloudiness. I'll check out molecular probes Thank you
>>> Gayle Callis 08/12/04 11:56AM >>>
We do a great deal of immunofluorescent (IFA) and GFP work for both
fluorescent and confocal laser scanning microscopes.
At 05:15 AM 8/12/2004, you wrote:
> I have several questions related to storage of slides stained for
> immunofluorescence. I do know they should be viewed as soon after
> staining as possible, but it is not always possible for our pathologist=20
> to do so.
> How long can they be stored without loss of reactivity?
**Preferably, look at them the same day and depends on what fluorophore you
are using. Some fluorophores will fade faster than others, FITC
photobleaches faster than Alexa 488. Rhodamines come in several
derivatives, rhodamine (TRITC) photobleaches faster than Rhodamine RX or
RRX from Jackson Immunoresearch. We now use Alexa fluorophores as much as
possible, due to less photobleaching plus extremely bright - or use a
Strepavidin Alexa conjugate with biotinylated primary or secondary.
****Hopefully IFA staining is done under cover of a dark towel or foiled
staining chamber and NEVER on an autostainer where you cannot protect
fluorophore from light. Don't start photobleaching before viewing or storage!
> Do you store them in the fridge?
**You can. We have stored them in slide cardboard folder, in dark and on
occasion in a freezer. The latter is not popular since slides must come
room temperature for viewing. We often look at them the next day, try not
to prolong this time too much - expensive in terms of time and material,
plus painful to lose fluorescent signal.
The biggest help is use antifade mounting media that reduces loss of
fluorescence aka photobleaching. Molecular Probes, Prolong Gold ready to
use is ideal for this purpose. It must be stored in -20C freezer with
dessicant, thaw (in my pocket!), use, then return to freezer after use,
but the benefits outweigh handling and fluorescence loss. You must seal
edges of coverslip with diluted permanent mounting media (dilute with
toluene or xylene to consistency of thin top coat nail polish, but do NOT
use nail polish itself.) This media is pricey, but worth its weight in
"gold". It sets up hard and since it is protective, sections can be viewed
again. Sections still need protection from all light - never on an open
slide tray. Go to www.probes.com/pl/prolong and look at the chart on
fluorophores with %loss using other media and Prolong Gold. It also works
with GFP nicely.
**Vector has VectaShield Hard Set that is supposed to work also. It is
nice to have an aqueous mounting media for fluorescent work that sets up
> After storage for several days, do you see more background, less
No more background than before, there are different reasons for background,=20
do you mean autofluorescence? or background from staining? If the
fluorophore photobleaches (fades) yes the autoflourescence background would
be more apparent. Preventing background due to crossreaction of
immunoglobulins to tissues, or aldehyde fixation is possible. Proper
blocking, dilutions, etc are part of this.
> After storage does your slide appear dirty when viewing microscopically?
**No, if you have a dirty looking slide (I do not know what you mean
exactly, particles that sit on a section and fluoresce? If the latter
happens (glowing garbage!) then spin a diluted fluorophore conjugated
antibody just before application and take aliquot off top and apply that to
section. Antibodies conjugated to fluorophores, even sitting around in
refrigerator OR when thawed, have protein aggregates (sp?) with fluorohore=20
attached, and this bright junk will sit around on top of section -
microcentrifuging briefly eliminates this problem.
If you do what we do, mount a coverslip on a slide taken from buffer, the
buffer salts sometimes leave a tidge cloudy film on back of slide. After
coverslipping, wipe back with kimwipe damp with water - takes off buffer
salts, all is clear.
Last suggestion, go to Molecular Probes and get their Handbook - a
wonderful, educational, free book on handling and storage of fluorophores,=20
and multitidbits on fluorescence related products. I believe you can
download it too.
Oh my, what a long discussion, been there - done it too many times
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
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