While we have had success with a goat anti-luciferase antibody(overnight incubation) from Promega followed by HRP conjugated anti-goat polymer and DAB, I cannot get any staining using the same luciferase antibody and a fluorescent secondary. I have tried increasing the concentrations of both the primary and fluorescent secondary and extending the incubation times and that only seems to bring up more background. I am working with mouse tissue so I prefer not to use a mouse primary.
Have you checked, if your reagens is still ok? Expired? wrong storage?
correct filters of the fluori-microscope ;-) ?
Maybe just too small amount of antibody. You can try to do an amplification-step in between the primary and the fluorescent secondary.
goat-primary - rabbit/anti goat - goat/anti rabbit - anti-goat-secondary fluorochrome.
Thanks Gudrun - All reagents are fresh, I am using the correct band-pass filter set. I think I will try some sort of amplification as you suggest.
Thanks Roxana - Yes, I am using donkey serum blocking (2-5%) before the primary incubation as well as in the diluent. The seondary incubation is a t room temp, but I can do it 37C and see what happens
I agree that amplification step(s) may be needed. I would suggest after the primary antibody incubation, incubate the section in SuperPicture Polymer HRP goat kit for 10 minutes (https://www.lifetechnologies.com/search/global/searchAction.action?query=superpicture&resultPage=1&resultsPerPage=15&autocomplete=), wash it, then incubate in TSA fluorescent system for 10 minutes (http://www.perkinelmer.com/Catalog/Family/ID/TSA%20Fluorescein%20System). Thereafter, DAPI and coverslip.
Well that's not exactly true if you noticed that I used an HRP conjugated anti-goat IgG polymer which provides some significant amplification by virtue of the numerous HRP moieties attached to that IgG molecule via the polymer. Whereas, I believe the fluorescent secondary has fewer conjugated fluorophores in comparison.
With our system we can easily perform multiple IHC staining (attachment of 4 marker staining), regardless of spices of primary antibodies.
-Hi Brett,
I also think amplification is needed. I suggest to use peroxidase conjugated secondary antibody, than biotine thyramide+h2o2 amplification step for 10 minutes, than streptavidnie and fluorescent molecule conjugated sec antobody.
I have also a quiestion: we try to make IHC on luc transgenic mouse in brain, we know that luc is there, but we cannot make any IHC, not even with DAB. Is there any trick in the protocol, which is essencial to catch this hard target?
Thanks, and good luck with experiments!
Petra
Dear Brett,
Why do you want to switch to immunofluorescence rather than Peroxidase staining? To obtain more signal, I suppose.
I want also yo know if you experimented your new antibody on the same sections that experimented for DAB.
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