I am trying to do double IHC-p/IF on mouse lung tissue. For the 1st antigen, Fas, I have a polyclonal rabbit-anti-mouse Fas primary antibody and a goat-anti-rabbit AF488 secondary antibody.
For the 2nd antigen, vimentin, I have a polyclonal goat-anti-mouse vimentin primary antibody and a donkey-anti-goat AF546 secondary antibody.
I have successfully done single stains with each of them separately, which roughly follows this protocol:
- Rehydrate from xylene to ethanol to ddH2O
- Antigen retrieval with Dako citrate buffer at 90 degrees C in hot water bath for 60 min
- For vimentin stain, I add in a permeabilization step here with 0.3% Triton X-100 in TBS-T at RT for 30 min
- Rinse 2x 5 min with TBS-T + 0.025% Triton X-100
- Block with TBS-T + 3% BSA + 1% serum (of secondary antibody host species) for 60 min
- Apply primary antibody diluted in blocking buffer and incubate overnight at 4 degrees C
- Rinse 2x 5 min with TBS-T + 0.025% Triton X-100
- Apply secondary antibody diluted in blocking buffer and incubate for 90 min at RT, keeping sections covered from now on to avoid photobleaching
- Rinse 3x 5 min with TBS-T
- Apply room temperature 0.2% Sudan Black B dissolved in 70% EtOH to sections to reduce autofluorescence, incubate for 15 min
- Rinse 2x 5 min with DI H2O
- Apply DAPI nuclear stain to sections and incubate for 15 min
- Rinse 1x 5 min with DI H2O
- Coverslip with glycerol diluted to 80% with TBS-T, seal with clear nail polish
- Allow approx 30 min to dry and then image
Again, single stains following this protocol have worked on their own. When I try to do double staining, I basically added in both primary antibodies during same overnight incubation, and then added in both secondary antibodies in same 90 min incubation, so the total time to do the protocol is the same.
Fas shows up great ... vimentin does not show up at all. Any ideas?
Thank you!
Dear Kelly,
I do think that the secondary antibody combination is not really wise choosen. Because it my happen, that your donkey anti goat will either mark the goat anti rabbit or that the secondary antibodies get inaktivated when mixed due to the fact that the donkey anti goat will bind the goat anti rabbit and since the antibodies can bind two they might cloumb and will not stain the trageted first antibodies.
Here the resolution:
Stain the primary together, and than stian first the donkey anti goat wash a few times and then stain with the goat anti rabbit. make sue with appropiate single staining controlls that there is no cross binding of i.e. the donkey anti goat to the Fas-antibody.
Best Soenke
Please let me know if that resolution helped.
Dear Kelly,
I do think that the secondary antibody combination is not really wise choosen. Because it my happen, that your donkey anti goat will either mark the goat anti rabbit or that the secondary antibodies get inaktivated when mixed due to the fact that the donkey anti goat will bind the goat anti rabbit and since the antibodies can bind two they might cloumb and will not stain the trageted first antibodies.
Here the resolution:
Stain the primary together, and than stian first the donkey anti goat wash a few times and then stain with the goat anti rabbit. make sue with appropiate single staining controlls that there is no cross binding of i.e. the donkey anti goat to the Fas-antibody.
Best Soenke
Please let me know if that resolution helped.
Kelly,
I have never tried to stain simultaneously, because there are often interferences between antibodies, both primary and secondary. (I have found it diffiicult to use multiple antibodies simultaneously even in western blotting, and never recommend it.) I recommend trying to do the 2 different stains sequentially. Another problem may be that the second antigen for which you are not getting signal requires a different or more stringent anitgen retrieval step.
I would first try to get signal from each antibody by staining separately, on separate slides to make sure that you can get staining from each antibody by itself with this retrieval method and the antibody concentrations you are using. Once you get both working, then try to do them sequentially, or at least, do the secondaries sequentially. And I would do the one that gives the best signal by itself first, because if you do the one with least signal first, then you may lose the signal in the extra washes.
Hope you find this helpful.
Karen M. Peterson
Thank you both for your input. Since this is immunofluorescence, I have been hesitant to try primaries sequentially because I worry that by the time I am done staining, the first antigen's signal will have faded significantly. My primary incubations have been overnight (although I suppose I could shorten them and try incubating at a warmer temp).
I will try the secondaries sequentially first ... thank you again!
I agree with the others: Try a combination of your primary antibodies over night, then wash. Then first your donkey-anti-goat secondary, rinse, then goat-anti-rabbit.
Your should not be afraid of loosing a bound antibody as long as they have a good affinity. And the fluorophores won't fade so quickly in normal room light, not within half a day! Especially not if you protect the slides from light during incubation and don't put then into direct sunlight. The fluorophores need kind of energy from light to be destroyed. While the strong light of the correct wavelength coming from your microscope can do this very quickly (seconds to minutes), the "broadband light" of your lamp bulb normally needs several hours to days or weeks for this, depending on the fluorophor.
That's reassuring, Vivica, thank you! It's going to be a few days before I can try it again, but I am looking forward to it!
Dear Kelly,
Try to avoid water in the end step ot sample rehydration. Also there is no need to permebilization at all - FFPE samples are already permeabilized. I think you should play with the epitope/antigen retrieval. Some epitopes do not withstand retrieval and are even destroyed. Second issue is the frluorescence but one can get to it in a later stage. Anyway after the last ethanol everything should be in a buffer ... in theory :)
Best regards,
Ivan
The suggestion to separate the secondary antibodies worked! Thank you all!
Ivan -- thank you for the note about permeabilization ... makes sense!
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