Hi Magda,

Did you used Triton before fixation? If your proteins are small they can "leak" out if the cell are permeabilised. 

The other possibility (less likely) is that your protocol affect the fluorescence of your probes. Fluorescent protein are sensitive to pH and if your buffer are completely off the neutral that could be a culprit. 

If you can add more details on your protocol that would help figuring out what is the problem.

Hope this help.

Max

Magda,

Triton is a strong detergent, that causes lysis of the cell membranes. If it's necessary in your protocol you may try to decrease the concentration of triton, dilute it. That should let to avoid excessive membrane permeabilization.

DMSO shouldn't cause fading or leaking out endogenous fluorescence, it's widely used as a solvent, also for fluorescent dyes.

I hope this will help.

Justyna

Does it look like there is a higher background to signal ratio? Or is there just less florescence in general? 

Do you happen to know the mechanism of the fluorescence of your stains? For example whether a prosthetic group such as a heme or porphyrins group is used for fluorescence or cyclization of some amino acids? 

In some cases, it is thought that the fixation process disrupts the β-barrel structure of the flourophores. Some flourophores, such as GFP, use three amino acid residues inside a larger β-barrel structure spontaneously post-translationally modifies itself in order to produce a fluorophore. These amino acids are sequentially Serine, Tyrosine, and Glycine. The Ser and Gly are thought to spontaneously cyclize via a nucleophilic attack by the Gly amino group on the carbonyl of the Ser residue. This five-membered ring spontaneously dehydrates to form an imidazole-like ring with delocalized electron structure. Following this, an oxidation of the linking group between the Tyr residue and the imidazole-like ring conjugates the two groups together to allow a larger delocalized electron cloud throughout the ring systems. The β-barrel surrounding the fluorescent group prevents exposure of the fluorophore to water and hydrogen bonds in a very specific manner that increases the tendency of the group to fluoresce, giving the protein a relatively high quantum yield. If the beta barrel is manipulated by the DMSO fixative there could be issues.

The chances of this being the case are small, since a protocol has been developed. But if you are using different tissues then the ones used in the protocol it is possible they you might need to switch your fixative, possibly paraformaldehyde or gluteraldehyde. 

Hello

I agree with all researchers

also ,for cells fixed with crosslinking aldehydes used reagents include DMSO and detergents like Triton X-100, saponin or deoxycholate . It is important to remember that every different specimen may require a different preparation method. Testing and optimizing each protocol for each new sample type will insure that the best balance between preservation and labeling is obtained. Therefore , shoud be add more details on your protocol that would help figuring out what is the problem .

Good Luck

Hello Magda:

I have used both of them in my staining protocols and nothing seemed to affect the fluorescence properties. But I use Triton at a very low concentration like 0.05%-0.1% just to make enough holes to help the probe/s enter the sites of binding. Also this incubation period should not last too long. Hope it helps.

Without knowing much about your preparation, its difficult to troubleshoot (although some is good feedback to think about above).  Triton can contribute to increased background fluorescence, particularly in tissues with a high lipid content or like brain or myelinated axons. You might take a look at the following paper where the authors examined exactly this question. Weruaga et al., 1998 Non-specific labeling of myelin with secondary antisera and high concentrations of triton X-100. J Histochem Cytochem 46:109-117.

   A potential way to address your problem might be to reinforce your GFP signal by immunolabeling with an anti-GFP antibody visualized with an FITC or AlexaFluor488 secondary antibody, which puts the endogenous fluorescence and the GFP immunolabeling in the same channel so that they are additive when viewing the specimen. There are a number of anti-GFP antibodies available. I know that Millipore and the Developmental Studies Hybridoma Bank sell them for certain.

A couple other things to think about:

  GFP fluorescence can bleach out. Depending on how much GFP you have in your cells and how you are observing it, the reduction in fluorescence could arise in part from photobleaching.

    Fixation, and other steps in a staining or immunolabeling procedure, can also affect fluorescent proteins. Long fixation times can reduce fluorescence (and antigenicity of many antigens). I would recommend against glutaraldehyde fixation which produces its own autofluorescent signal (paraformadehyde does not have this problem to the same extent). 

     I would also look into whether DMSO can affect GFP fluorescence. GFP fluorescence is diminished by dehydration when embedding tissue for plastic sections. I have no first hand knowledge of any DMSO effects, but it seems like something that could contribute.