Re: [Histonet] Long discussion on Immunofluorescence/slide storage

From:Kathleen Roberts

Here's a question: has anybody tried those Quantum Dot antibody or 
streptavidin conjugates?  Supposedly this stuff DOESN'T  fade, ever.  
I'm looking into it, but have not tried them yet., in case anybody is interested.

Kathleen Roberts
Principal Lab Technician
Rutgers University

Gayle Callis wrote:

> 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 
> 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
> 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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