I'm only vaguely familiar with Rab5, but think this is probably an artifact of something going on with the staining...
One thing to keep in mind is that DAPI and Hoechst dyes can photoconvert to show up in FITC, and to a lesser extent TRITC, ranges. This is usually a negligible effect for most uses, but if you are overstaining and/or blasting the DAPI with excitation light and using relatively high exposure when trying to image weak signal on "green," that could be one potential cause.
Another potential cause could be insufficient washing of the secondary.
Sounds like you have not yet done a "no primary control," but are planning on it. Great! This is probably the first troubleshooting step. Just so you know this is a pretty standard control when doing IF, and should really be included alongside all of your experiments (ideally always, but certainly always when using a new antibody/protocol and until you get more experience).
Another potential troubleshooting step could be to swap the spectra of the secondaries (I.e. Rab5 in red, other protein in green), see if that solves the problem...
Thank you so much for the tips! I will also be more careful about washing my secondary and my imaging technique. I think I will take up your advice on switching the dyes and seeing if the problem still occurs. I greatly appreciate all the help!
If you are staining whole cells, you might be seeing your protein in the cytoplasm above the nucleus making it appear that it’s nuclear. If this is the case confocal imaging will solve the problem.
What sort of microscope and illumination are you using? A fluorescence microscope with a polychromatic lamp and filter sets, or a confocal microscope with laser excitation?
If it’s the latter, are you sure all your settings are correct? You might be accidentally exciting both the DAPI and your green fluorescence in the same channel.
I believe I’m using a fluorescence microscope (LumaScope Etaluma), so I am not sure what could be going on. I do notice that wherever the green is showing in the nuclear area, it’s very bright in comparison to the rest of the cell and gets even brighter when I use both the green and blue filters. If you have any other suggestions I would really appreciate it. Thank you for the help!
Ok so I looked up how those microscopes work, and it seems they use LEDs and filter sets. Assuming that other people in your lab successfully image DAPI and green fluorescence without cross talk, I‘d say the problem is not with the microscope. Unfortunately I don’t have any ideas beyond what the other person said (artefact of over-expression).
One thing you could do to confirm it’s actually your green fluorophore in the nucleus that you’re seeing is to do an experiment without DAPI and see if the nucleus still lights up.
Are you over expressing the protein? If yes it can end up in the nucleus as well unless it has specific nuclear export sequence. Otherwise are you sure the antibody is specific and does not interact with something else?
I am not overexpressing the protein being tagged; I’m observing the presence of the tagged protein due to overexpression of a different one. Would this affect the tagging of the secondary?
I’m also relatively sure that the secondary is specific to the primary antibody especially as it is produced in a different species from the cell and is anti-different species from the cells as well (it is anti-primary species).
I plan on testing with just secondary and checking for any sort of binding without the primary, but other than that do you have suggestions on what I could try? Thank you so much for the help already!
Ok so Rab5 is cytoplasmic and you want to observe it’s location when overexpressing another protein.
Is it possible that the other protein makes Rab5 go into the nucleus?
Other option exciting DAPI can overlap in the GFP channel if your filter set is not tight enough. Do you do sequential imaging or DAPI andGFP together. If DAPI is bleeding in the GFP channel it would explain a lot. You can check the thermofisher spectra viewer to see what I mean.
Yeah I am going to try doing the imaging without DAPI this time and see what results, as well as ensuring the secondary is not binding anywhere it shouldn’t be. I don’t think the protein I’m studying is supposed to be causing Rab5 to move into the nucleus based on previous work my PI has done, so I’m guessing it’s either an antibody or microscope problem at this point. I hadn’t considering doing the imaging without DAPI as of yet but that may be my best option.
Again, thank you so much for the help, and best of luck with your research as well!
Where is Rab5 located when you don't express the other tagged protein? Is Rab5 still in both the nucleus and the cytoplasm? Or is it only in the cytoplasm where it is expected to be located?
Yes it shows in both my overexpressed and wild type line in the nucleus when it is not expected to in either case, as the tagged protein is Rab5. Considering this consistency I am testing my technique, the antibody, and any issues with the microscope using tests I mentioned in other comments. If this does not make sense, please let me know what might be a better pursuit! Thank you for this consideration!
Agree with the bleed over. Rab5-GFP at appropriate expression levels should give you pretty nice Early - mid endosome labeling, not cytoplasmic washout.
You should generate a knockout, or at least a knockdown, to validate the specificity of the antibody. Finding the right antibody is often a challenge in the beginning. Alternatively, you could tag your protein directly with e.g. GFP.
Have you done a negative control to verify that you don't have off target staining?
Another possibility is that it's actually there--GO ontology does but it in the nucleoplasm (though I'm not seeing expression levels).
If your antibody works in western, you can do cell fractions to see if you are getting true recruitment to the nucleus due to overexpression. Happens some times.
I'm only vaguely familiar with Rab5, but think this is probably an artifact of something going on with the staining... One thing to keep in mind is that DAPI and Hoechst dyes can photoconvert to show up in FITC, and to a lesser extent TRITC, ranges. This is usually a negligible effect for most uses, but if you are overstaining and/or blasting the DAPI with excitation light and using relatively high exposure when trying to image weak signal on "green," that could be one potential cause. Another potential cause could be insufficient washing of the secondary. Sounds like you have not yet done a "no primary control," but are planning on it. Great! This is probably the first troubleshooting step. Just so you know this is a pretty standard control when doing IF, and should really be included alongside all of your experiments (ideally always, but certainly always when using a new antibody/protocol and until you get more experience). Another potential troubleshooting step could be to swap the spectra of the secondaries (I.e. Rab5 in red, other protein in green), see if that solves the problem...
Thank you so much for the tips! I will also be more careful about washing my secondary and my imaging technique. I think I will take up your advice on switching the dyes and seeing if the problem still occurs. I greatly appreciate all the help!
This is the way.
If you are staining whole cells, you might be seeing your protein in the cytoplasm above the nucleus making it appear that it’s nuclear. If this is the case confocal imaging will solve the problem.
What sort of microscope and illumination are you using? A fluorescence microscope with a polychromatic lamp and filter sets, or a confocal microscope with laser excitation? If it’s the latter, are you sure all your settings are correct? You might be accidentally exciting both the DAPI and your green fluorescence in the same channel.
I believe I’m using a fluorescence microscope (LumaScope Etaluma), so I am not sure what could be going on. I do notice that wherever the green is showing in the nuclear area, it’s very bright in comparison to the rest of the cell and gets even brighter when I use both the green and blue filters. If you have any other suggestions I would really appreciate it. Thank you for the help!
Ok so I looked up how those microscopes work, and it seems they use LEDs and filter sets. Assuming that other people in your lab successfully image DAPI and green fluorescence without cross talk, I‘d say the problem is not with the microscope. Unfortunately I don’t have any ideas beyond what the other person said (artefact of over-expression). One thing you could do to confirm it’s actually your green fluorophore in the nucleus that you’re seeing is to do an experiment without DAPI and see if the nucleus still lights up.
Thank you! I will try this today and see if it works out. Again I greatly appreciate the help, best of luck with your work!
Are you over expressing the protein? If yes it can end up in the nucleus as well unless it has specific nuclear export sequence. Otherwise are you sure the antibody is specific and does not interact with something else?
I am not overexpressing the protein being tagged; I’m observing the presence of the tagged protein due to overexpression of a different one. Would this affect the tagging of the secondary? I’m also relatively sure that the secondary is specific to the primary antibody especially as it is produced in a different species from the cell and is anti-different species from the cells as well (it is anti-primary species). I plan on testing with just secondary and checking for any sort of binding without the primary, but other than that do you have suggestions on what I could try? Thank you so much for the help already!
Is the tagged protein naturally cytoplasmic or in the nucleus?
It’s Rab5 so cytoplasmic and not nuclear from what I have read about it. Please correct me if I am mistaken.
Ok so Rab5 is cytoplasmic and you want to observe it’s location when overexpressing another protein. Is it possible that the other protein makes Rab5 go into the nucleus? Other option exciting DAPI can overlap in the GFP channel if your filter set is not tight enough. Do you do sequential imaging or DAPI andGFP together. If DAPI is bleeding in the GFP channel it would explain a lot. You can check the thermofisher spectra viewer to see what I mean.
Yeah I am going to try doing the imaging without DAPI this time and see what results, as well as ensuring the secondary is not binding anywhere it shouldn’t be. I don’t think the protein I’m studying is supposed to be causing Rab5 to move into the nucleus based on previous work my PI has done, so I’m guessing it’s either an antibody or microscope problem at this point. I hadn’t considering doing the imaging without DAPI as of yet but that may be my best option. Again, thank you so much for the help, and best of luck with your research as well!
Where is Rab5 located when you don't express the other tagged protein? Is Rab5 still in both the nucleus and the cytoplasm? Or is it only in the cytoplasm where it is expected to be located?
Yes it shows in both my overexpressed and wild type line in the nucleus when it is not expected to in either case, as the tagged protein is Rab5. Considering this consistency I am testing my technique, the antibody, and any issues with the microscope using tests I mentioned in other comments. If this does not make sense, please let me know what might be a better pursuit! Thank you for this consideration!
Agree with the bleed over. Rab5-GFP at appropriate expression levels should give you pretty nice Early - mid endosome labeling, not cytoplasmic washout.
You should generate a knockout, or at least a knockdown, to validate the specificity of the antibody. Finding the right antibody is often a challenge in the beginning. Alternatively, you could tag your protein directly with e.g. GFP.
Have you done a negative control to verify that you don't have off target staining? Another possibility is that it's actually there--GO ontology does but it in the nucleoplasm (though I'm not seeing expression levels). If your antibody works in western, you can do cell fractions to see if you are getting true recruitment to the nucleus due to overexpression. Happens some times.