How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

Question

**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](https://en.wikipedia.org/wiki/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](http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/fluorophores-and-their-amine-reactive-derivatives/alexa-fluor-dyes-spanning-the-visible-and-infrared-spectrum.html?icid=fr-alexa-3#head1), and [pHrodo Red](https://www.lifetechnologies.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2012/bioprobes-68-november-2012/phrodo-ph-indicators.html), 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?

Answer 1

In general, no, this should not really affect the pH of the lysosomes. When you are fixing the cells with a crosslinking agent such as formaldehyde, you're only immobilizing the proteins while maintaining the structural integrity of the protein for the most part. Sometimes, you may be masking the epitope that your AB recognizes during fixation so this is important to keep in mind.

With the detection issues you are facing, I am immediately drawn to your choice of fluorophore. While IHC is something I do sparingly, I do a lot of flow cytometry which is another fluorescence-dependent assay. Just a slight correction, Alexa-488 isn't the brightest Alexa-fluor available, and relative to truly bright fluorophores such as PE or APC, I would say it is only moderately bright at best with optimization. Have you tried using a different fluorophore-conjugated antibody ? When dealing with lowly expressed proteins, it is ideal to use the brightest possible fluorophore available to aide in detection. Additionally, assuming you are using a confocal microscope, are you filter sets optimized to detect fluorescence in the realm of 515 nm ? In my experience when facing issues with fluorescence detection, I've been able to solve my problems by changing the conjugated fluorophore, or optimizing my detection method (ie. changing filter sets, using a different laser of excitation). I hope this offers some help.