[Histonet] Nail polish seal vs diluted permanent mounting media) Why's, another freebie
Yes, Permount or similar permanent media. Just use the same solvent e.g.
toluene or xylene already contained in media. Nail polish contains
isopropyl alcohol which leaches into aqueous mounting media under coverslip
and/or PBS (we have used this). Nail polish avoidance is reported in
literature (Science, GFP publication) Toluene and xylene are NOT water
miscible, don't leach into aqueous medias under coverslip. Alcohol causes
GFP protein and maybe other fluorophores(?) to not fluoresce. Another
mounting media reported in literature that works (not sure how much
photobleaching prevention it is capable of) is Mowiol. Molecular Probes
has a comparison chart of several aqueous mounting medias showing which are
superior for preserving fluorescence - very enlightening.
Molecular Probes suggests paraffin sealing, NOT fun, ultra-messy, and if
you use inverted fluorescent microscope with confocal set up or other wise,
objectives get slimed with paraffin! MP said to avoid nail polish with
Prolong Gold, but is may be because people use this for GFP
work. Clontech (GFP gurus) suggests using rubber cement (ooey gooey messy
too!), and suggested solvent diluted permanent mounting media.
Another hint on free literature. Clontech website, go to literature and
download Living Colours Manual -this freebie has tons of info on GFP,
mounting media, fixation, and autofluorescence.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
>In terms of sealing the coverslips with permanent mounting media--do you
>mean something like permount? Also, can you explain why you advise NOT
>using nail polish itself??
>Morphology Core Manager
>Buck Institute for Age Research
>8001 Redwood Blvd.
>Novato, CA 94945
>From: Gayle Callis [mailto:firstname.lastname@example.org]
>Sent: Thursday, August 12, 2004 8:56 AM
>To: Mildred Fail; Histonet@lists.utsouthwestern.edu
>Subject: [Histonet] Long discussion on Immunofluorescence/slide storage
>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
> > 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
> > background?
>No more background than before, there are different reasons for background,
>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
>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,
>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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