Definitely go for 0.1% paraphenylene diamine in 50-90% glycerol in either PBS or better 100 mM Tris with pH between 8-9. Best way to prepare it is to bubble argon through the solution before adding glycerol, as PDA is not readily soluble in aqueous media, or alternatively dissolve the intended amount of PDA to VERY LITTLE DMSO and once dissoloved mix it rapidly with the rest. Note that you need to be fast and gentle, NOT introduce oxygen to the solution (it oxidizes very easily and you can tell that by seeing it turning black overtime) and store it in dark, -20o or even -80o

Dear Didier,

I always used Vectashield and Fluoromount mounting media with excellent results for my immunofluorescence stainings.

One frequent cause of your problem could be that you allow your cells to dry during the staining (for example when you wash), so you have a uniform background in all the sample. But the background could be also a problem of the staining, for example a too much high concentration of antibodies or insufficient washings, or maybe a problem of your sample, for example the fixation and permeabilization of the cells. Another thing to consider is if you are using reagents incompatible with each other: in my experience, when I performed immunofluorescence stainings on tissue sections and I treated them with Sudan Black B (to reduce autofluorescence of the tissue) I could not use the Vectashield as mounting medium because this latter is not compatible with SBB, and the background was so high that I was not able to detect any signal!! In this case I used 70% Glycerol in PBS.

Good luck!

I would also try to use more concentrated blocking buffer. Usulay I use 5% BSA but if it is necessary I apply up to 8%. It could also be problem with slides. Maybe try other manufacturer. Good luck.

I used the fluoromount from sigma, which gives little background when polymerized. In my experience, waiting some days or one week before taking photos is better.

DAKO Fluorescent Mounting Media . Used for fluorescence and confocal and never had problem. Hope this helps you! Good luck!

Hi,

Flouromount from Sigma is good but yes most of the times if IF is done without storage, Glycerol 70-90% in PBS works good. Best thing is anything like a GFP which ahs a tendency to give lot of background with storage should not be mounted with glycerol, not so stable. Alexa's will be ok with any method. Just that your storage and sealing should not allow cells to dry up, enough of mounting medium should be used.

Dried and shrunken cells give background through auto fluorescence. Storage of coverslips in 4 degrees in darkness is fine for weeks.

After testing a large number of aquaous mountant, i recommand Fluoprep, a french product that i use systematically in all Immunofluoresce techniques. You should find micrographs in my publications. Cheeers.

did you ever try to omit the antifade reagents. some of them really have effects on the efficiency and brightness of certain fluorophores. prepare a sample without cells and see if the background is persistent - if no, something with your dyes/staining procedure is wrong - OR your microscopy.

josef

I was satisfied with Vectashield and Fluoromount mounting media. Both provided very good results and both for my immunofluorescence staining and DiI tracing. But be careful: you should leave your embedded slides in the dark for hardening before fluorescent microscopy and photography. Visualization of fresh slides will provide fast fading.

We have used DAKO Fluorescent Mounting Media for years and have never had a problem. Its an excellent anti-fade formulation.

cheers,

Jeff

By far the best anti-fading agent is para-phenylene diamine (PDA). PDA is a little tricky to prepare as it is easily oxidized but when you prepare it correctly you get very stable immunofluorescent preparations. Briefly: Rapidly dissolve a small amount of PDA flakes (roughly 0.01 g) in a minimal amount of DMSO (30 microliters should be fine for a 10 ml batch of antifade mountant) taking care to minimize contact with air. Immediately add 1 ml of 1 M Tris-HCl pH 8.4 and then 9 ml of absolute glycerol and mix with a glass rod adequately. Aliquot the mixture in 1.5 ml eppis and store to feezer (-20oC) or deep freezer (-80oC). The mixture blackens with time so if it turns too dark then discard. Solid PDA is sold by Sigma (P6001) and it can be stored at room temp or better at 4oC.

I used prolong gold antifade reagent with dapi from Molecular probes by life technologies. Works really good. they have with dapi and without. ( cat # P36935)

I used 2.5% DABCO and it worked exceptional for IF as well as confocal...

Hi Didier,

All of the products you have tried ought to be giving you satisfactory results with many of the common fluorophores, so I am wondering if there is something in your protocol creating this problem that is NOT the choice of mountant. Can you explain in more detail exactly how you handle the samples? Also, are you using a counterstain and if so, what is the protocol and type that you use? Do you mount wet from PBS or water, or are you drying or dehydrating? What are the stains? What is the imaging system? etc.

hi everyone,

so many reply!

we are doing as many of you are doing..

fixation/extraction: PFA /0.5%tx/PHEM1X 20min

saturation/blocking :30min 3%BSA

antibody diluted in PBS/1-3%BSA/0.1%tx 1h

and so on..

I have since the original post try several mouting media

and my best results is with prolong antifade...let dry ovn and sealed the CS...I have observed that the signal is more stable this way (indeed, this is what lifetech advice!)

I will post images, I have time

cheers

I like ProLong too, I use it most often. Best to let it dry flat overnight and then seal the edges with nailpolish-it has a refractive index that changes after dru=ying and gives better results after a day or two than when used fresh.

My experience is that mounting mediums work differently with different secondary antibodies. I have had very good results with Alexa Fluor 488, Red-X, and Texas Red using 0.5% N-propyl gallate, 90% Glycerol, 20mM Tris pH 8.0. I can not comment on how well it works with the Cy secondaries.

For IF experiments I prepare my own mounting medium with Glycerol and 2X PBS to make a 70% Glycerol solution. I use them mostly for temporary slides. I have used mounting medium from Invitrogen with DAPI and that worked fine. For permanent slides with DAB/Silver stained slides Permount works best for me.

I prefer using 50% Glycerol (with coverglass sealed with VALAP) for temporary slides and Apathy's mounting medium for long storage. To lower photodamage and increase storage time I use Oxyrase.

Can any one can tell how you to do best monting

(1) Put 20uL mounting media and the put coverslide

(2) pusg to remove the excess mounting media wit hpaper (buth this will creat air bubbles)

I have too much blue background (might be from dapi) i use mounting media with dapi....

can some one please help me???????????????

I usually place the cover slip on a piece of filter paper, then add 10 uL mounting medium in a drop on the coverslip and finally, I place the slide, upside-down on top of the coverslip.

The weight of the slide will be sufficient to remove the excess water, and I avoid any excess force on the coverslip. Thus I seldom have problems with bubles.

Concerning the background fluorescence... Sometimes I do see a little excess blue fluorescence, but the difference between background and the DAPI signals should be suffieciently big that you reliably can remove it by reducing exposure time when obtaining images. - Could you perhaps post an image, so that I can see the degree of background compared to specific nucleic stain?

I have noticed, that when to high a concentration of DAPI (or hoechst 33342) is used, this might also give signals in the green channels - thus if the concentration is a problem, you might be able to see the nuclei in the green channel as well.

Thanks casper, i controm the background with lss exposition. But normally i it should not be soo much background..... i will upload a photto soom....

I've been doing this for 25+ years, and we only mount with gelvatol, a home-made mounting medium. Extremely low background fluorescence/autofluorescence, excellent for GFP fluorescence. See the recipe here:

http://www.cbi.pitt.edu/protocols/gelvatol.htm

Irfan: Mounting medias with DAPI in them always give more background. It is far better to stain with DAPI then rinse about 2 x 2 min in PBS and then mount with a good aqueous anti fading mounting media. You will be happier with the results.

Irfan shaukat!! you can use Prolong Gold antifade reagent with DAPI (Molecular Probes). It retards both auto-fluorescence as well time dependent fading of fluorochrome conjugated antibodies.

Autofluorescence can only be omitted if you are examining your slides towards far infra-red range. The likelihood of getting autofluorescence is more at UV and visible range, Also it depends on which tissue you are using. In your case the blue slide is due to the excess of DAPI concentration. If you dont want to use antifluorescence media- then dilute your DAPI 50X and 100X and see the results.

In my experience, background appears if you let your preparations get dry at any step in the course of staining...Avoid drying the slides while you are transferring them from fixative to the buffer.

The simplest is Prolong Gold from Invitrogen.  You can get it either with DAPI or without DAPI.  Cat # P36941.  Always works.

I do not work with cryostat sections, and my technique concerns fixed permeabilized cells. Normally I mount my cells (I use total preparations of ciliates) in 60-70% glycerol in PBS containing Moviol, according to the manufacturer protocol. This mounting medium becomes hard, and there is no need of sealing the edges with nail polish, and it is a good antifade. I store the slides in the fridge at -20, however, fluorescence intensity strongly depends on the quality of the fluorochrome - sometimes keeping at room T does not affect fluorescence, sometimes it does. I prefer not to keep the slides for too long and have a session at the microscope the next day. However, sometimes antibodies and fluorochromes are quite stable.

It can be hard to predict the level of quencing. 

With some fluorophores I have managed to have decent results after several weeks storage at 4 degrees celcius without using any antifade. 

However recently, when I have been working with FISH, I have noticed significant quencing overnight in the fluorescent intensity. Here an antifade agent were necessary. However, here I had to observe small fluorescent dots within the nuclei, meaning that even a small degree of fading would be significant.

I would suspect, that if your signal is strong at the moment, and small details are not needed, the signals should be fine for at least a week at, when you are storring the samples at 4 degrees. I have not experience with storing at room temperature, but lacking any antifade reagent and storing at room temperature is risky. I would be interested to know the result.

Amira, fixed sample could be store for a long like. Our lab sill has a sample of mouse liver  tissue with Alexa 488 Fluor from 2012, which we often use as a demo for our new students...

I have also never had a problem with DAKO fluorescence mountain medium. It's quick and easy to use and provide good results.

http://www.dako.com/us/ar38/p107570/prod_products.htm

As you are using Rhodamine and not one of the newer anti-fading dyes like the similar Alexa 568, you can expect more rapid quenching. Using Alexa fluors, cold/dark storage,  and a good anti-fade like ProLong Gold or Dako, I have successfully imaged for several months. Using FITC or Rhodamine with no preservative in the medium,  the dyes can fade before my eyes under the microscope.

I make my own mounting media for my IF staining which is nothing but 80%glycerol-PBS. It is not very thick and not very diluted. It works just fine and I can take images next day.

Moviol is best mounting media...

What is the concentration of DAPI to be added to the gelvatol protocol?

We prefer a combination of far-red DRAQ5, with incubation for 30 mins. and tamping of excess, followed by either Fluoromount-G or ProLong Gold. A clinical lab I have visited strongly prefer Fluoromount-G following an extensive side-by-side study on different mountants. 

Try using Fluoromount-G.

Fluoromount-G is for me the best for IF using confocal

Fluoromount-G with DAPI is the best assembly medium I have tried and since then (5 years ago) it is the one I use for my IF. It saves you the step of DAPI counterstaining and covers very well all kinds of preparations.

Vecta shield with DAPI staining separately, works fine for ICC.

Ranu Pal

How much parts from Glycerol and PBS you have taken to prepare the mixture and what if I have Glycerol 86%!

https://nic.med.harvard.edu/resources/media/

Depending on what you intend to do, the answer will vary. If you are looking to quantify the images, it is important to consider the refractive index. Fluoromount has a RI of 1.40 while prolong antifade has a refactive index of 1.52.

You should match the RI of the mounting solution with the immersion oil. The RI of the oil and cover slip are about 1.51 so it is a good idea to consider antifade over fluoromount.

Mounting solution can be purchased commercially or prepared in the laboratory

We use the following custom mixture. To 90 mL glycerol, add 10 mL

of 10X PBS that had been adjusted to pH 9 with 0.5 M Na2CO3. Dissolve n-propyl

gallate in this solution to 5% (w/v) using bath sonication. Store at −80°C in 200-μL

aliquots.Cited by Methods Mol Biol.

I have been using

PVA mounting medium with DABCO from Merck #10981 for the last 15yrs, and take DAPI separately. Works fine and is less expensive. If it thickens in the bottle just heat it 5-10min at approx 60°C. It is important that you wait some hours with microscoping the slides. S/N ratio then improves for several mounting media.

Our lab use Vectashield mounting medium with DAPI for IF/ICC experiments.