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Microscopy and Imaging Techniques: Fluorescence and Immunofluorescence
 
Kristie Nybo, Ph.D.
BioTechniques, Vol. 46, No. 6, May 2009, pp. 403–405
Full Text (PDF)

This month's questions from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) come from the “Microscopy and Imaging Techniques” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.

Molecular Biology Techniques Q&A

Can I detect an immunolabel without GFP interference? (Thread 21952)

Q1 I need to perform immunofluorescence on cells and am required to use a green fluorophore. My cells, unfortunately, will be expressing GFP as an shRNA reporter gene, but I'm not interested in that GFP signal and it will interfere with signal quantification of the immunofluroescence. Is there any way that I can quench the GFP fluorescence to practically zero prior to immunostaining? Will this negatively impact the immunostaining?

A1 It would be a lot easier to just use another fluorophore or a YFP signal instead. Is it possible to try that?

You could try photobleaching the cells before immunostaining, but I think that they might recover during the time you complete the immunostaining. You could look into crystal violet, but make sure to check if this will quench your fluorophore as well.

Q2 I think photobleaching might work, and if it was done after fixation, it would probably stick. But I've never tried it and I'm not sure how much light would be required.

The reason I would like to use a green immunolabel even though my cells express GFP is because I obtained my shRNA plasmid through collaboration with a core facility that will make viruses for transduction and do other favors, provided that I use this plasmid. It expresses the shRNA to my protein of interest, but also expresses a GFP reporter for transfection efficiency. While that's nice, my ability to see this GFP reporter isn't really that important.

In my assay, I'd really like to be able to stain with four colors: DAPI plus three immuno-labels. We're set up to do DAPI plus green, red, and far red fluorophores in the same sample. I don't think that I can avoid using the green with our instrumentation. I suppose I could eliminate the GFP by some cloning, but I'd rather avoid that if there's a simpler way around it.

A1 In my lab, we've had a much harder time trying to maintain GFP fluorescence than lose it. Our normal fixation protocol with paraformaldehyde and acetic acid doesn't keep the GFP photoactive. We visualize GFP via immunofluorescence with an anti-GFP antibody. If we want to view the GFP directly in fixed cells, we have to use a different fixation procedure without acid, but with the addition of methanol.

Just try fixing a GFP+ sample and look in the green channel. I doubt there will be any signal.

A2 Strong reducing agents such as FeSO4 can convert GFP to a non-fluorescent form. Fluorescence can be restored by exposure to oxygen, so be careful with the samples after treatment.

Can I detect both GFP fluorescence and an immunolabel simultaneously? (Thread 17398)

Q1 I want to transfect my cells with some GFP/YFP containing constructs to label cellular organelles. After transfection, I will infect the cells with a virus and fix the cells at a certain time point after infection. To detect my virus, I will need to do indirect immunofluorescence. Is it possible to detect both the GFP (without staining) and my virus (via immunostaining) at the same time?

My protocol for immunostaining asks me to dehydrate the slides after PFA fixation. Will that affect the GFP signals?

A1 You can do both fluorescent and immunofluorescent microscopy, as long as the secondary antibody you use is labeled with a fluorochrome that needs a different filter than GFP/YFP. If you use the same filter, the two signals won't be distinguishable.

With regard to GFP quenching, I do not know the conditions that decrease the GFP signal, but I think it is caused by additional fixation by either ethanol or methanol.

A2 We use an antibody to GFP when we do our immunofluorescence. Just co-stain with two different antibodies: one for GFP and one for your protein of interest.

A3 Use paraformaldehyde, not methanol, to fix your cells or you will lose your GFP signal. I recently had this exact problem and found the answer on another thread on this forum.

A4 I know that GFP does poorly when exposed to acetone and it is very sensitive to dehydration. I suggest that you fix the cells in some sort of formaldehyde. I used both formalin and glutaraldehyde with great success. Never let GFP dehydrate since it really diminishes the signal.

Here is a fixative solution that I have used in the past that works well: 2.7 mL 37% formaldehyde, 0.4 mL 25% glutaraldehyde, and 46.9 mL PBS. I leave it on for 5 min at 4°C, rinse with PBS, and store the slides in PBS.

By the way, all of this applies to native fluorescence. You can use any fixative you want to if you are going to do immunocytochemistry with an anti-GFP antibody.

Did you check the living cells for a signal before you fixed them? You should look at them if possible to be sure that they are expressing the protein.

A5 GFP photobleaches rather easily, so keep that in mind when you excite it. Also, why not transfect with enhanced GFP so there won't be a need for an anti-GFP antibody?

When you stain for your virus, try to use a far red secondary antibody such as Alexa 633. Texas Red is okay to use, but it bleeds into the FITC channel where you will view the GFP. This will be a concern if you are looking for co-localization. If not, then Texas Red is okay.

A6 GFP is a protein that has to maintain a specific fold to achieve fluorescence. If you want to keep the signal, don't use anything in the immunocytochemistry protocol that unfolds proteins.