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Immunofluorescence troubleshooting - (Sep/22/2011 )

Hello all! I'm a longtime lurker, first-time poster, and I was hoping you guys could help me out a bit.

I recently tried immunofluorescence and got the staining to be very nice several times (back in June). One day, though, it failed on me, and I haven't gotten it to work since! Basically, after significant analysis of my protocol, I have found a few differences between what my lab normally does and what standard procedures dictate.

1) Fixation is carried out for 30 minutes, as opposed to 10-15 recommended
2) Permeabilization is carried out over 3 10-minute washes with 0.6% triton-PBS

What I have ended up with recently is very high background and poor nuclear staining with DAPI. I took a look at the pH of my paraformaldehyde, prepared yesterday, and found it to be near 10.

The questions I am getting to are as follows:

1) Can the cells be "overfixed," and what is the consequence? How will the higher-than-recommended pH affect the overall result?

2) Can the cells be over-permeabilized?

I must say, today was better than others. Before I could not focus on my protein through the coverslip, and the background was too high to catch any signal by eye (my software is able to deconvolute somewhat, but it's still garbage results). Today, I had high background but could at least pick out my fluorescent staining of focal adhesions. Still, though, no nuclear staining whatsoever.

Any tips or referrals to good troubleshooting sources would be very much appreciated! Thank you very much!


What are your samples (cells, tissue(and type), clots...)? What percent formaldehyde are you using? Please give more specifics on your protocol - such as incubation times, washes, antibody concentrations etc.

You can certainly overfix, it will crosslink the proteins tightly, which may affect antibody binding.

You can't over-permeablise really, this usually just punches holes in the membranes and helps dissociate some proteins.

High background may be because of autofluorescence or some residual formaldehyde, which fluoresces a greenish-yellow.


Thanks for the reply. Here is the information you requested:

3.5% PFA, 30 minute incubation then wash three times with PBS. Then 0.6% Triton X-100, 3 times ten minutes each, then blocking with 3% BSA for an hour before overnight incubation of the antibodies.

The cells are MDA-MB-231 cells which have been seeded on fibronectin and laminin for approximately 4 hours to allow them to attach, spread, and start migrating.

Perhaps today I will try a 15-minute PFA fixation and make sure to pH it carefully.


15 minutes should be fine. I usually do 10 minutes in 2% PFA at room temperature for cells. I then wash 6-10 times in PBS and PBS with 0.1 M glycine (mops up free PFA) before permeablising.

0.1% triton in PBS for 10 minutes should be enough permeablisation.


Thanks for the reply again, Bob. After 15 minutes permeabilization and using carefully-prepared PFA and checked the autofluorescence before adding secondary (minimal background at that stage). I used a previously-used mixture of antibodies that gave me very good staining some time ago along with the most recent mixture that gave the high background.

This time was better than before but not as good as what I was hoping for. A few observations:

1) DAPI staining was present but very poor, at times if you could even see it it would look like material was leaking out of the nucleus, staining as a sort of smear in the blue channel

2) High background was still present, but it showed that background was not due to GFP (which is expressed in these particular cells) but to the antibody

3) The staining I used was FAK pY397, which stains the focal adhesions very well, giving sharp, punctate signal. In this case with my older antibody mixture I could see that staining but it looked fuzzy, as if somebody used the blur tool on my nice, sharp staining.

I spoke with my boss about it, and he took a look at the corresponding brightfield that I took with the background fluorescence and said with certainty that the permeabilization had been inadequate, and the primary and secondary antibodies were, as a result, not able to be washed out of the cell adequately. This seems suspect to me, since we used 0.6% triton in our all our washes and all our antibody solutions. Also, considering the poor DAPI staining, it seems to point to a different problem. He told me that immunofluorescence has been done for decades and that we shouldn't waste our time repeating (I suspect that 0.6% is too much and that we should try with a lower concentration, which is what prompted that)

So, to summarize the protocol I used:
1) Grow MDA-MB-231 cells on fibronectin/laminin-coated coverslips
2) Wash with PBS and fix with 3.5% PFA 15 minutes, followed by 3 washes with PBS
3) Wash 3x 10 minutes each with 0.6% Triton in PBS (prepared by adding 1.5 mL 100% triton to 250 mL PBS) to permeabilize
4) Block with 3% BSA in the triton buffer
5) Incubate overnight with primary antibody
6) Wash 3 times; incubate 1 hr RT with secondary
7) Wash, mount, visualize

I am quite dismayed at this result, since it's another one of those protocols that should just work, according to the boss, and there is some error I am introducing. As you can imagine, my confidence has taken a hit. Can anybody provide some further guidance? I greatly appreciate it!


Is this the same protocol that you used when you had the staining working well? If so, perhaps your antibodies are going off, which can happen occasionally. It could be that you aren't washing enough either, though your times seem fine.

My standard protocol is:
Grow cells on coverslips
wash in PBS 1x
Fix 2% PFA, 10 min at room temp.
Wash many times in PBS
Permeabilise 10 min in 0.1% triton x-100
Block in 3% BSA in PBS with 0.5% tween-20 (PBS-T)
Dilute primaries in block, incubate for required time
Wash 3-4 x in PBS-T, 5-10 min each
Dilute secondaries in block, incubate
Add DAPI in PBS-T, 10 min incubation
Wash 3X PBS-T as above

DNA outside the nucleus is a bit unusual; have you tested your cells for mycoplasma recently?


Note everyone who may have the same problem, look into the simple things first! I was growing cells on plastic coverslips. Yes, I feel like a moron, but at least I now know the ins and outs of IF. Thanks for the advice!