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?