I'm looking for solutions to maximise the signal-to-noise ratio in immunofluorescence and/or FISH. We are routinely using Alexa Fluor labelled secondary antibodies but now we need something more powerful. We primarily work with formalin fixed tissues, so the color should preferably NOT be green (due to autofluorescence).
Dear Mikael,
I would recommend a tyramide detection kit (TSA), which you can use either to get a FITC or TRITC (or other fluorochromes) signal. See for example here http://www.perkinelmer.de/product/tsa-tmr-reagent-pack-100-300-slides-sat702001ea, here http://www.emu.uct.ac.za/sites/default/files/image_tool/images/430/lecture-4-intracellular-localisation/tyramide.pdf, and here https://www.perkinelmer.com/lab-solutions/resources/docs/MAN_TSA_Plus_Fluorescence_Manual_V1-0.pdf.
It is, however, quite expensive compared to fluorochrome conjugated antibodies.
The signal is enhanced by the activity of HRP, and fluorochromes precipitate near the bound antibody. I had good experiences using FITC-tyramide for FISH. The system is commonly used for both immunofluorescence and FISH.
Thanks! I have previously successfully used home-made biotinylated tyramide in immunohistochemistry, but I haven't really tried fluorescence. Perhaps I should. The drawback in tyramide amplification is that it utilizes peroxidase, and the endogenous (pseudo)peroxidases might generate some background. I'm looking for maximal signal-to-noise ratio so this is not ideal, but perhaps still useful.
Ok, then you should try to get rid of the endogenous peroxidases before you perform the in situ / immunofluorescence. did you try bleaching with H2O2 (http://www.ihcworld.com/_technical_tips/peroxidase_tips.htm)?
Yes sure, it helps but it's not perfect. I know tyramide amplification is good (and happy to hear that it works well for fluorescence as well!) but it's not perfect. Might be good enough anyway!
As a target is typically bound by several antibodies, it might help to enlarge your "detction sandwich". For example, if you have a human-anti-XYZ antibody as primary ab, use an unlabelled rabbit-anti-human in the second step and a labelled goat-anti-rabbit tertiary antibody for detection. It might help, but might also result in more background signal as all existing signals get multiplied.
If you already used biotinylated tyramide with success before, the easiest (and cheapest) solution would be to use that in combination with a fluorescent streptavidin (coupled with a red or far red fluorophore, to avoid the green autofluorescence). You should have less background like that, compared to a direct Fluorophore-tyramide amplification, provided that you don't have too many endogenous biotin in your tissue. Otherwise, Cy3-tyramide or Cy5-tyramide kits from Perkin Elmer should work as well.
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