I believe it depends on your fixation method, and the mounting medium that you use. I think it also depends on the sensitivity of what you're doing. If you're just taking pictures you'll probably be ok, but if you are doing careful quantification work, and comparing the old samples to recently processed samples - that may not be possible without bias.

Hi Hosam, Tony is right, depends on fixation and mounting. I use FFPE tissue and mount with Fluoromount G (also ProLong medium is very good), which is a aqueous-based medium that keeps fluorescence for a really long time. If you stain sections with quality Alexa Fluor or cyanine-conjugated antibodies you can re-check your slides for months. They do not fade at all (at least by eye). If you just need to take another picture there will be no problem at all. Of course if you want to quantify fluorescence intensities (FI) you must never mix old and new samples. The only way you may do this is comparing relative FI, lets say measuring your marker of interest and relativize to an element in the tissue or cell that stains homogeneously and reproducible, that of course may have faded in the same way as your marker of interest.

Personnally, I have 3-years old samples and I checked it recently and already is ok.

But Tony and Maria have right, it's depending of your samples and your acquisition.

Hi Hosam, I agree with both Tony and Maria that your fixation and mounting methods are important in determining how long the fluorescent signal lasts. Most of the mounting solutions I've used give a signal for around 1-6 months but I usually prefer to repeat the IF.

Hi Hosam, I agree with previous comment..all depends on fixation and mounting medium (I have used ProLong on tissue fixed with PFA 4% and it lasted more than a month). To add to this, also if you "close" your slides, by putting nail polishing on the sides and sealing it, it slows down excahnge with air and thus prolonging fluorescence. And again, more importantly, if you have to quantify fluorescence, you cannot use "old" sample (the use of an internal standard, in my view, it's not so correct..you never know if the FI decades on the same level as the protein of interest), but unfortunatly you have to make new...hope this helps :)

It also depends on how long you were looking at them the first time - if it was for an extended period of time (like when acquiring high resolution z stacks) that can cause the samples to bleach out. Otherwise, with optimal fixation and mounting, you should definitely be okay for a couple of months!

Both Maria n Tony are right.... While mounting media vary n everyone claims superiority of their own, they are all fairly good. So it's gonna boil down to ur fixation... But as u say, in the interest of time and resources, u may wanna check ur slides quickly for fluorescence signal n compare to ur previous images of the same slides to assess preservation of ur specimens...

Indeed, it depends on what are your needs and the fixing/mounting protocol. Of course, when trying to compare fluorescence intensities, you should do it only from the same set of samples. But if you just need a qualitative result (is it there or not), then more often than not it will be still fine also after a couple of months or even longer. I used fixing with PFA and mounting with Mowiol 4-88, we also usually used Alexa dyes, and the stability was not an issue, although I wouldn't be able to tell now how old were the oldest samples I've viewed. My supervisor even said I could store those slides in freezer, but I don't think I ever looked at those ones.

Why don't you just have a sneak peak at those slides and see if they're any good? See if using the same confocal settings you still get a comparable intensity as before, if the cells look still fine, if there's any background issues, and then you can see if you need a new set of samples or not.

I agree with the previous answers. As an example, i have HDMEC that have been fixed with 1%pfa for 30 min, stained with Alexa488, and mounted using prolong gold. I have re-imaged these over a year later and still obtained great quality pictures. As mentioned, it would be wrong to compare these to previous images, but you could redo the imaging of these samples.

In addition to the above, if possible after labeling your specimens with fluorophores, re-fix the tissues in paraformaldehyde... this prevents any dissociation that might occur and retains the fluorescent signal at the original binding site. Works really well with Alexa dyes

The fixation and refixation ideas are crucial, but an important issue for slides stored at 4oC is diffusion despite fixation. I'd be leery of any slides kept in the refrigerator - a non-cycling freezer if far preferable.

I agree with the previous answers, that stressed the importance of fixation and mounting medium. I prefere hardening mediums, such as moviol and vectsshield. But you should be careful: you can not put your slides ander the microscope at least during first 6-7 hours after coverslipping. Keep them in the dark box at room temperature till next day and only after that replace to the refrigerator. Another moment - importance of your sectioning. Vibratome sections are faded slower than cryocut ones.

Your best bet is to include an antioxidant into the mounting medium and seal the cover slip. Photobleaching/fluorescence fading is mainly caused by ROS. Also of importance is to hermetically seal your coverslip to prevent the slides form drying out. Under these cicumstances you can keep and view your slides for months

Regarding FI relativization I meant you can relativize to the FI of an element in the tissue that stains homogeneously and reproducible for the SAME marker. Of course, this do not occur very often... Relativization to another marker, as Fulvio said, it is no really correct because different proteins/dyes may decay in a different rate. In my experience I personally found quite accepted internal relativization to the very same marker of interest expressed by other cells in the same tissue. Being the same marker (labeled in different cells) it must decay the same way. Me for example I have worked with fetal human tissue which is very hard to get regularly. I got samples every month if lucky so the stainings for a given marker differed in months between them. My protein of interest (being a highly conserved protein) was luckily expressed in three different cell types in the same tissue, and one of those cell types expressed it homogeneously and reproducible (fact supported by observation and by the literature). Relativizing the "problem" FI to the FI of those cells was the only way I had to compare FI from all my samples colected during months.

As the other, all come down to the fixation method and mounting medium you are using! Personally, a classic PFA 4% with Alexa dyes should last quite a bit of time. Please be sure if you are using a non drying mounting medium you have to seal your cover slip completely otherwise condensation will ruin your slides. I used a home made Mowiol as mounting medium and check some fluo slide 2-3 months after without noticeable change! Good luck with your experiments.

In my experience under those conditions 2-3 months. All the best

I fix mammalian cells with 3.7 % Formaldehyde and mount with aqeous medium containing anti-fading agent. Under those conditions, the signal (alexa fluores) is stable for at one month. Good luck

Usually, I fix mammalian cells with 3.7 % Formaldehyde and mount using aqueous medium with anti-fading agent. Under these conditions, the signal (alexa fluores) is stable for at least one month. Good luck

I agree with all comments on very long stability of Alexa dyes at 4°C in the dark, with a good antifading product. The new Dylights (488 and other wavelengths) secondary antibodies are as good as the alexas : intensity and stability. In case you have stained your nuclei, look at your slides in the 2 weeks coming because DAPI fade quiet quickly. A

Fab

Thank you all fellows for your valuable feedback. That was quite helpful and informative.

I am not sure that FI relativization is the best approach (when it comes to quantification), unless one is sure that the unspecific fluorescence intensity decays at the same rate in the two samples. One way passed this (theoretically as I didn't test such an approach) is to relativize to a test beans of the stain prepared at the same time with the specimens and stored under the same conditions. Still this wouldn't be completely accurate as the signal decay is not only due to changes in the dye but also in the substrate.

It is really an interesting issue and I really hope that someone will test the different suggestions given in this discussion and share their findings.

Yepp, if you fixed your samples well, and have them in Vectashield or Fluoromount G, sealed it all nicely in (nail polish does the trick nicely), and kept them in a cool and dark place, you should be fine imaging them for half a year or so. I see fading at 1-2 years, not months. It's the imaging itself that can bleach your Alexa signal fast if you use high laser power in a confocal.

Hosam,

Panc-1 cells are good cells to work with. As other users have mentioned, as long as your samples have been through Vectashield they should be fine. Alexa's 488 staining is of great quality, again no problem there. Be careful though when you take them out for additional imaging. Let the samples acclimatize for 10 min at RT and then be careful and smart with your laser source, the prolonged exposure can be detrimental. Many years ago I used to store slides that I wanted to come back again at some point in a period of 6 months, wrapped in foil, placed in slide holders and stored at -20C. I dont know if other users still utilize the latter trick.

Good luck,

Georgios

Depends on your fixation protocol and the protein what you have stained. Transmembrane proteins need

While I know the mainstream does not do this, I have used rapid dehydration through alcohols then xylenes and coverslip with low fluorescent mounting media. If well dehydrated, the sections for most fluorophores can last for years and years with no fading (Cy2, Texas Red, Alexa orange, or Cy3) Only if you do not allow the slide to completely dry after coverslipping have I seen fading. This approach is idea for those fixatives that contain acrolein in addition to formaldehyde.

Hosam, it seems you already received the right answer - depending on tissue, preservation of it, fixation, embedding, thickness of the slides, buffer, atb / primary and 2ndary, simple or biotinylated, mounting you can get less than 3 days to more than 3 months good preservation - just look at them: intensity / fade, edge effect, cleannes of the slide, absence of crystals, precipitates, foggy areas.

Good luck,

Andy C

I usually fix mammalian cells with 4% paraformaldehyde, mount in Aqua-Poly/Mount Coverslipping Medium and seal edges of coverslips with nail polish. I see fading after 1-2 years. If your slides are quite freshly made, try to seal edges of coverslips with nail polish, it should stop bleaching.

Don't use too high laser power in confocal mode, the fluorescence of Alaxa dye will come back to previous state.

Hi,

I work on invertebrate frozen section fixed in 1% or 3% paraformaldehyde-PBS, after the first observation, and I usually keep them at -20°C. I can reobserved the slides with a good signal even after 6-8 months.

Good luck

Hi,

I usually fix mammalian cells with cold methanol, mount in PBS and usually keep them at- 20°C, good signal after 2-3 months.

It depends, it should be fine for a month. Next time if you like please keep them in -20 degree

I usually fix mammalian cells with 4% paraformaldehyde and stored them at -20 °C, good signal after 6 months.

It is no safe criterion to predict how long reaction will stay. Recently I found in the fridge an old box with slides of mine from last year visit in the Lab DPAG Uni of Oxford. And they showed reaction. Not the best, but could take some pictures. So usually, when you need them to stay fluorescent they don't, when you don't need, fluorescence is still present.

It depends on the fixation and mounting protocol. I usually fix animal cells or sections with 4% PFA in PBS or methanol. For embedding I normally use an embedding medium containing glycerol and N-propyl-gallate (anti-fading reagent). With this protocol the fluorescence will stay relatively constant for several months.