[Histonet] Long discussion on Immunofluorescence/slide storage

[Gayle Callis]

Rena,

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 
hard!!

>   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

Gayle Callis
MT,HT,HTL(ASCP)
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)