We would like to immunostain mCherry on sections of our transgenic zebrafish. To detect mCherry we use the abcam antibody. We tried on paraffin and frozen sections, but we didn't have any immunoreactive cells.
What I would like to find is something I can use as a positive control for the mCherry immunostaining to determine if the absence of immunoreaction on our samples comes from the antibody or from the fact that our transgenic fish do not express the transgene.
Can you see the mCherry signal in live fish using fluorescence?
As for positive control, maybe you should get in a couple known mCherry-expressing transgenic fish at a couple different expression levels to use as a standard curve.
We probably need to know a little bit more about your detection system. I am guessing that you are using indirect immunofluorescent detection of a protein.
If that is that is the carse, it is possible that your primary and/or secondary antibodies have not been optimized or have gone bad. You may need to optimize the concentration of your primary antibody and for testing purposes change the secondary antibody to a different color that your system can still detect. You could also switch the primary antibody to a known target that works in the zebra fish to test your mCherry detection antibody.
There can be many questions here..
Did the antibody (AB) work before? Sometimes they go bad. (freeze thaw etc.)
Some AB are good for western but not for IF. (was the AB used for western and you are trying it for IF?)
if you have mcherry cells in culture try the AB on them with green fluorophore and compare localization.
The best positive control would be a case in which you can still see the mcherry fluorescence. Then compare IF with green or another color.
How do you know its mcherry? I recently got cells from a collaborator who said they were mCherry, but there were dsRed (the plasmid was mislabeled on their end) I could tell by the fluorescence lifetime that it was not mcherry.
Thanks to all.
I will try to answer everyone.
We received our transgenic fish recently and we are trying to characterize them. In our transgenic fish, using a macroscope and a filter set specific for mCherry we see a little bit of direct fluorescence in the tissues of interest at the expected localization. But since it also fluoresce a little bit in green (detection with a GFP filter set) at the same localization I was wondering if what I see could be autofluorescence of the tissues. So I tried immunohistochemistry.
But when we tried to detect this fluorescence by immunohistochemistry with a specific anti-mcherry primary antibody we found nothing.
For these IHC experiments we used alexafluor488 and 594 as secondary antibodies and our microscope is equiped for the detection of these AB (filter set for "rodhamine": excitation BP550/25 and emission BP 605/70; filter set for GFP: excitation BP470/40 and emission BP 525/50). We performed all our previous IHC with these secondary AB and never had any problems.
Concerning the antibodies, we tried different dilutions of the primary and secondary antibodies.
But it is the first time we use the primary AB against mCherry so I don't have any idea if it works well. This AB is sell for WB, ICC/IF and IHC-P so it should work (it is the abcam AB). We added glycerol to avoid freezing problems with it.
My probleme is that I don't have any sure positive control to know if this AB could work with our protocol of IHC. And We don't have any mCherry cells in our lab to test this AB.
And to answer a last question, normally fish should have been transfected with a mCherry-reporter gene even if mistakes could occur. But we also obtained from the same firm other transgenic fish with GFP-reporter gene and we had no problems with them.
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