The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes?

Some background: I'm trying to look at the intracellular trafficking of a fluorescently-labeled lysosomal enzyme. Cells are grown in vitro and treated with the labeled protein, which is taken up into the cells via receptor-mediated endocytosis. The protein-receptor complex targets the late endosome-lysosome pathway, where eventually the acidic pH of the lysosome causes the two to dissociate and the receptor to recycle, while the enzyme stays and does its thing. I've labeled the enzyme with two different dyes - Alexa Fluor 488, which is supposedly pH-insensitive between 4 and 10, and pHrodo Red, which has greatly-increased fluorescence as the pH drops. Alexa488 is the brightest of the Alexa dyes, and is widely used in all sorts of applications. pHrodo is not as bright (as far as I can tell, it's been difficult to find comparisons), but its pH-dependence helps lower the effect of non-specific extracellular binding, and it seems to work in my system so far.

So what's the problem? I'm developing an assay that will measure the ability of certain substances to block the uptake of this enzyme into the cell. Since I'll be measuring a lack of signal, the higher I can get the uninhibited signal to be, the better. Like I said, pHrodo works OK, but it could be better. The problem is that my Alexa-labeled protein has an extremely low signal to noise ratio, when I would have expected the exact opposite. I'm wondering if, in my system, the Alexa dye really is somewhat pH-sensitive, and is not fluorescing as well when in the acidic lysosome. I'm encountering a few other issues as well, and while before I was performing live-cell imaging, I'm starting to toy with the idea of fixing the cells first, as that will positively impact some other things I want to try.

Hence my question: Will fixing the cells with 4% formaldehyde in PBS (standard ICC/IF protocol) affect the pH of the lysosomes? Would methanol fixation do the same thing? Any other suggestions for increasing signal?

  • $\begingroup$ i don't have a definitive answer.. fixation can also affect the dyes.. formaldehyde can react with the fluorophore and make it insensitive. pH should change on fixation because it dehydrates the sample. $\endgroup$
    Sep 19, 2013 at 6:52
  • $\begingroup$ Alas :) I see, I suggest that fixing with formaldehyde will be best, but the buffering itself will affect the pH of the system, but you're better off. Also I'm curious as to whether formaldehyde will crosslink and "plug" ion channels in the lysosomes, thus stabilizing the internal pH... As mentioned above, this will be time-dependent, so you could try fixing with formaldehyde and monitor the pH of lysosomes with pH sensitive dyes and see if changes through time... could be a quick/neat experiment.. $\endgroup$
    – user4739
    Oct 23, 2013 at 13:44

2 Answers 2


In my opinion, cell fixation shouldn't change the pH. However unbuffered formalin will oxidize and lower the pH, but using PBS should buffer around pH 7. Maybe Glutaraldehyde fixation would also be an option, if the others are not working...

I found this website by leica very useful, maybe it will also help you:



Yes, they will both affect pH.

In addition to the above comment, both, but methanol especially dehydrates samples - removal of water will most definitely affect the pH in organelles. This will be time dependent. Both duration of fixation, and time after fixation before you assess the cells will affect the pH of the organelles.

Because you fix the cells in a non-aqueous environment (I presume you're using standard fixation), you don't use buffer during fixation. We might be able to answer better if you post the general protocol you follow.

  • $\begingroup$ thanks for answering! I actually fix with 4% formaldehyde in PBS, so it is an aqueous environment, as I mentioned in the question. $\endgroup$
    – MattDMo
    Oct 23, 2013 at 2:23

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