that is in principle fine, but the point is to keep the pH in all the solutions you use physiological, because at the heart of the GFP the proton making the fluorophor is pH dependent...

PFA 2% for 15 minutes... good luck !!

PFA can work well with GFP, but some folks find PFA tricky to make and use. If it's freshly made, RT or cooler, and you pH'ed it carefully, then it's likely fine. Also be sure to keep your processing time short and not expose your sample to much light - room fluorescence can kill a gfp signal pretty fast, so try covering your preps with foil while they incubate. Cold methanol is also a good fix for gfp; you might give that a go to see if there's a difference. If you have to, try an anti-gfp antibody with a complimentary secondary to bring up your signal.

I had very good results after perfusing the mice with 4% paraformaldehyde followed by the standard sucrose infiltration and then freezing for cryosectioning. Without perfusing with 4% PFA, the GFP has not been visible...

I currently use PFA 2% for 30min but 15min could work as well!

4% is fine but really depends on what you are doing. Kusumi, (Nature Methods 7, 865–866 (2010)) tested many protocols for fixing single GFPs. It should help...

ah, right, and it depends whether you destroy structures - if you want to see nuclear morphologies an MAA fixation is a no go...

2% PFA for 15-20 mins at room temperature seems to work well for me. If you still have weak signal (it may not be the fixation method, just not very much GFP!) then try an anti-GFP antibody to amplify the signal.

PFA 4% should be fine, but the signal may vary. If it is weak you might want to consider using an anti-GFP antibody to increase the signal.

I have had very good success with 3.7% PFA for 5-10 minutes. Just make sure that it is relatively fresh and that the pH is 7.4. I normally do a 10 minute wash with warm physiologic saline or HBSS prior to the fixation to wash serum components off of cells. Regardless, I have found that from person to person, and lab to lab some tinkering with wash time and fixation time is needed.

Hi Mark,

2 or 4% PFA should be ok. Do you see a significant difference between the signal pre-fix and post-fix? If your signal needs a boost you could consider labeling with an anti-GFP antibody.

4% PFA is fine, but only if you have it buffered to pH=7, eg in PBS. If the pH is too far away from 7, proteins will denature and GFP is no exception. I used to fix over night with 4% formaldehde when I wanted to go home in the evening, and the GFP was still fine the next morning.

Did you compare to signals without fixation?

Did you use some antifade reagent to avoid quick bleaching?

Did you use high NA Objectives, oil or water immersion to maximize brightness?

Hi Marc, your fixation protocol sounds suitable to me, I never had problems when I fixed my cells this way. I wondered whether you had a checked your cells under the fluorescence microscope when they were still alive? How much signal did you loose upon fixation (rough estimate)?

I agree with Monica and Steffen 4% PFA is fine for me but pH is important.Alternatively have you tried in vivo imaging?

Hi Mark,

Fixation with (para)formaldehyde is suitable but make sure you rehydrate your cells for a few hours (e.g. with PBS). Also, I'd recommend using a fluorescence mounting medium such as Vectashield (Vector Laboratories) or Fluoroshield (Sigma, F6182).

It may be worth checking for expression levels with a quick western blot and an antibody against your protein or GFP first.

Good luck!

PFA (paraformaldehyde) fixation preserves GFP signal in both, Tetrahymena and mammalian cells. MEOH fixation in -20oC does not (checked on mammalian cells).

Tetrahymena cells -

depend upon localization of GFP (cytoplasm, proteins bind to MTs and so on) you can

1. first fix with 2% PFA (we make it in PHEM buffer to stabilize microtubules) and then permeabilize with triton-X-100 (we use 0.5%, also in PHEM).

2. Permeabilize / fix with mixture of 2% PFA / 0.5% Triton (1:1) ratio

3. or first permeabilize and next fix

I used to use 3.7% fresh PFA in PBS to fix the cells. Now I've been using 4% and it works pretty well too. Before fixation, I recommend to rinse the cells with warm PBS or HBSS (with Ca++ and Mg++) twice, then fix them in room temp for 15 min. Also I would cover the dishes with foil to avoid day light bleaching (not sure if it's the big issue, but it might help to reserve the fluorescence). Gook luck!

2% - 4% PFA (EM grade) for 10 min is fine, but you must avoid any organic solutions, such as methanol, EtOH, or acetone. These reagents will kill the fluorescence,. If you need to permeabilize, us triton or saponin.

Steffen Dietzel · 31.04 · 180.62 · Ludwig-Maximilian-University of Munich presents a way to preserve with a good routine which always have good results

We use 4% PFA in PBS, 30min for cells, or via perfusion for mice. If you find it's not bright enough, I recommend using an anti-GFP antibody (the one from Aves Labs, made in chickrn, is excellent)

GFP is quite tolerant of fixation conditions. I've used formaldehyde, paraformaldehyde (4%-8%) in varying phosphate buffers and still see signal, from 5minutes - 20minutes (but not any longer).

Weak GFP signal is usually due to the actual amount of tagged protein that is stably expressed. So either:

1) increase expression; or:

2) use an anti-GFP antibody, either directly conjugated to a fluorophore or you can use fluorescent secondaries.

As suggested above, do check if the protein is stable when tagged with GFP.

As long as you don't use methanol or acetone GFP will be fine. Formalin or PFA work best for fluorescent proteins.

Good luck

Hi Mark, I agree normal formaldehyde fix should be ok for GFP but I came across this protocol a while ago, its formaldehyde but slightly different procedure, its meant to be better for preserving GFP fluorescence:

Remove media from cells & wash once with warm (37°C) PBS

Fix in (0.3% triton X-100, 4% paraformaldehyde in PBS pre-warmed to 37°C) for 7.5 minutes room temperature.

Remove fix I and wash cells with 2 rapid exchanges of PBS

Add fixative II (4% paraformaldehyde in PBS), for 7.5 minutes room temperature

Wash with PBS (RT) 5 minutes x 3

The fixing should be done on ice with 4% paraformaldehyde (also I make the 4%PFA in PBS). 20 mints fixation also is not a problem. Try floromount G for mounting the coverslips on the glass slide. 20 ul of floromount G is sufficient for each coverslip. After which you can image the cells a day later. Always keep the slides away from light whenever possible. Keep the slides in a black box in 4 C away from light when not in use. The slides is properly stored is good for 3 weeks and even more. But try to get the images within the first two weeks.

Regards,

Somasish

I agree with people have been saying here. 4% PFA should not be a problem, and if you're fixing simple flat, cultured cells, 10-15 minutes at RT is enough. However, if it is a piece of tissue for a whole mount for example, you should extend it to 20-30 minutes, so the tissue is evenly fixed. Methanol is good, but it can be quite dehydrating and generate artifacts such as protein clustering. Of course, always wash your cells with PBS before fixing, and never let them dry.

So first, fix and do the whole process always protected from light, put your dish/plate in the drawer, for example. Mounting can be quite critical, so I highly recommend prolong antifade. Lastly, you can always add an anti-GFP antibody if your reaction settings allow you to add an extra one.

Good luck

Felipe

I have had good luck with methanol, unlike most other answers here. However, I primarily use it on C. elegans and fix at -20C. Methanol will precipitate soluble proteins, however, so it is more appropriate for membrane proteins or any other fluorescent fusion that adheres to cellular structures.

Paraformaldehyde works very well for fixing GFP in position, even in the cytosol, though it can increase autofluorescence which may be a problem in cells that express very low levels of GFP.

I use 4% PFA/0.1M PB on HEK cells for 15 min, not any longer. I recommend the Aves chicken anti GFP antibody too. Mount with prolong antifade. I still have a good fluorescence signal 6 months later.

Fixation in 4% PFA is good. Try mounting your sample in 1 drop of DAKO fluorescent medium / sample, directly post fixation to preserve GFP signal.

The pH can be critical, so try to keep it a bit high (see: http://www.sciencedirect.com/science/article/pii/S0006349598778701). I use pH 7.5.

May be for this reason a good mounting medium is also really helpful, we use Vectashield.

About fixation, we use PFA4% for very long periods (up to weeks) at 4ºC with plant tissues, no problem.

If GFP is all you're looking at, there is no need to fix the cells at all. Depending on your microscope, you may not even need to take the medium off, so you can look at your live cells over time. Also, no need to fix for flow cytometry IF GFP and maybe PI is all you're looking for.

PFA is fine; PFA followed by methanol (5-10 min) iworks as well as does PFA followed by mild detergent, while methanol alone does not work. I prefer PFA/methanol to PFA/Triton as the former treatment also flattens the cells and (depending on the cell type) can improve the optics so that widefield fluorescent microscopy works as well as does confocal. Also consider that there are a huge range of different GFPs available now, which have mutations aimed at improving their stability and brightness. My data are based on eGFP (Clontech) and eYFP.

hi everyone, i need to ask sth depending on the answers discussed above. Are all of these suggestions for cell lines or tissue sections ? which procedure do you recommend for HeLa cells transfected with a GFP tagged protein ? does the prolonged fixation with PFA (1h) affect the signal negatively ?

We use 4% PFA in PBS for bacterial cells expressing GFP or mCherry, works great.

I tried again this week with ice cold 1% PFA. Again the signal eas very low. One thing to note was I also had easily detectable punctate auto fluorescence in the peri-nuclear region (in pcDNA empty transfected cells). A lot of people here say 4% PFA is good but many others on other forums say this kills most of the GFP. I am wondering if it is fine if you have lots of GFP expression (10% of a lot is still quite a lot) but not good if you only have low expression (10% of a small amount is not much). Maybe I should try the anti GFP antibody approach.

Thanks for all the advice.

Mark

Mark,

Proper pH of fixative and good mounting media is the essential for a good signal to noise ratio. You may want to make up fresh 4% (or less) PFA, which can be stored at 4 degrees Celsius for a week or so.

For a 4% PFA solution follow this protocol.

Add 4 grams of PFA to approximately 70mL of water

Heat up to 60 degrees in a water bath (do not heat any higher)

Add a few drops of 1M NaOH until the PFA goes into solution

Cool to room temperature and add 10mL of 10x PBS.

Adjust the pH to 7.4 and bring final volume up to 100mL

Fix cells for 15-30 minutes, wash away fix with 5 volumes of PBS before putting cells into blocking/permeablization step. Also, make sure your blocking step has glycine in it to properly quench the PFA reaction.

Gulden,

This if for tissue culture cells. 1 hour of fixation is overkill in most circumstances I believe. You should have better luck with 15 - 30 minutes of fixation.

Hi Mark, a really interesting question and a lot of neat ideas, particularly paying attention to the pH. I would also add: 1) Making sure that the osomolarity of the PFA solution is physiological (e.g. 16% commercial solutions are sometimes made in water, so you have to adjust for the volume of water while diluting in buffer). 2) Using warmed PFA. Cold PFA might mess up membrane fluidity, and possibly cause GFP leakage before complete crosslinking of the soluble GFP to the intracellular matrix has occured, not to mention slowing down the crosslinking reaction - Kiran

In my hands the best has been PLP fixation, which consists in 1 % PFA with L-Lysine and sodium periodate to quench autofluorescence for 24 hrs followed by an overnight incubation with 30 % sucrose. After this treatment, you can snap-freeze your tissue in OCT and store until making frozen sections. Good Luck!

I would recommend formalin fixation (we buy ready to use formalin from Sigma Aldrich) for 15 min and depending on the specific other antibodies that you might want to use for co-staining then additionally 0.2 % Triton permeabilization for 5 min. WE used it standardly and we get good fluorescence data on GFP cells. In general when fixing GFP cells it is important to avoid any reagents that could potentially quench the signal.

Hi Mark,

I guess that it also depen on your downstream application. For a few pics, a short fixation with a well-buffered, freshly made 4%PFA in PBS might be enough (although there will be some loss of intensity). If your aim is serial confocal reconstruction, you better go for an anti-GFP coupled to an alexa488 (similar spectrum) and mount with anti-fading. The amplification will drown the autofluorecence. I hope this helps

may be the problem is not fixation, I have used both formaldehyde 3.7% in PBS and 2 or 4% PFA (more or less fresh), of transfected cells or cells expressing stably (or inducible) EGFP/mCherry/mRFP tagged protein, for epifluorescence, confocal imaging, FRET etc..

If you are imaging your samples immediatly or a few days later there is no problem, if you are keeping the samples what you do after fixing might be more important, for instance use anti-bleach agent or mounting medium with antibleach, like moewiol/DABCO (in this case you need to wait 24 hours before observing your samples). I have also kept fixed cells in PB (best) or PBS in slides with microwell,with enough liquid, kept tightly closed with parafilm and protected from the light with foil at 4oC I was even able to transport the slides and image them else-where.

2 % PFA (more or less fresh) for 30min work goog for mamalian transfected cell with GFP construct... We use vectashiel as mounting medium, and we observe samples few days after the fixation and it's works well! Good luck!

In my hands fluorescence signal decreases with longer fixation time. So I would lower as much as possible the fixation step.

For Bacterial cells, 0.25-1%PFA  fixation for 20min is effective in our lab. I was using such fixation for fused and free FPs in Microscopy and Fluorometry analysis and the result is highly consistent. 

Hey Marc, Your fixation protocol seems to be OK. It should work well.

I think you should take the suggestions given by Timothy. They are awesome.

Mark Bond, do you found a solution to observe GFP-cells after fixation? I have this problem too and wanted to resolve it rapidly.

Thanks a lot.

hi guys,

it is all about keeping the pH constant, because the fluorophore within the GFP is pH dependent and can be even completely destroyed - thus also avoid everything which is changing the structure of proteins, fix but do not alter anything else...

also always prepare the fixative fresh !

for a guide see here:

Article Trichostatin A-induced histone acetylation causes decondensa...

or here:

Article Construct conversions caused by simultaneous co-transfection...

I always fix with 4% PFA made fresh right before every use from a 16% PFA stock and D-PBS. Like you, I incubate for 10min at room temp., wash once with D-PBS - from here I will either store the cells in D-PBS or 70% EtOH (depending on the future use I may do all this as RNase/DNase free).

I haven’t had a problem with this method, I use both transfected & non-transfected cells (mainly endothelial) and they are fixed for IF and FISH. For IF and FISH, I grow the cells on coverslips inside 12 well plates- this is what I store them in until I am ready to stain.

Hi, Mark Bond!

We always use 4% paraformaldehyde (in PBS) to fix cells for immunofluorescence at room temperature for 20min or 4 ℃ overnight. 10 minutes in 4% PFA is OK for fixation. I think there are other problems, such as transfection efficiency or the abundance of protein tagged by GFP.

Here's the transfection protocol.

Hope this would help you.

Hi, Mark Bond!

We always use 4% paraformaldehyde (in PBS) to fix cells for immunofluorescence at room temperature for 20min or 4 ℃ overnight. 10 minutes in 4% PFA is OK for fixation. I think there are other problems, such as transfection efficiency or the abundance of protein tagged by GFP.

We launched a product of Lipogene, which is comparable to lipofectamine 3000 but with fewer side effect. You can find the protocol of this product attached.

Hope this would help you.

Hello,PFA defientely decreases the GFP signal. It has been happening to me always. My transfection efficiency was 100% and then after IF when I check the cells under conforcal it seems to Be like 50% transfection efficiency.The only way to enhance your signal is to use GFP antibodies. I have done this once And the difference was significant.

best of luck

In my experience GFP is not really denatured during Fixation. At least not for 4% PFA if prepared fresh, like Viviana Volta