Hello, everybody.
I want to perform IHC with my tissue sample. I have the antibodies for the markers that I want to see, but all of them are conjugated with the dye such as FITC, PE or APC. Can anyone tell me whether IHC is possible with these fluorescence-conjugated antibodies? Is it just OK if I use the secondary antibodies on these primary antibodies?
Yes you can use the appropriate secondary and it will bind to your primary. May be some parts of the primary won't be accessible to the secondary due the conjugated marker, but as long as the secondary is a policlonal antibody it shouldn't be a big issue.
You might also consider a different approach using a primary antibody against the labeling marker (i.e. anti-peroxidase).
Yes you can use the appropriate secondary and it will bind to your primary. May be some parts of the primary won't be accessible to the secondary due the conjugated marker, but as long as the secondary is a policlonal antibody it shouldn't be a big issue.
You might also consider a different approach using a primary antibody against the labeling marker (i.e. anti-peroxidase).
There are also antifluorescein antibodies conjugate with biotin that can be used.
Here is a example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584034/
The tags on these antibodies should also work as an antigen which you could detect with the appropriate antibody. We tag primary antibodies with FITC so that we detect that primary with a biotinylated secondary. See responses from Chaves and Rotilant above.
Find out the nature of your Flourescinated (FITC, PE) conjugated primary antibodies. Are they F(ab)2 or whole IgG; Then find out whether these FITC/PE primaries are derived monoclonally or polyclonaly? If they are monoclonal F(ab) I would consider them better. However it is polyclonal F(ab) you may have good immunostaining. Second concern is why should you be using 2nd antibody after using FITC/PE primaries? Where do you want the such 2nd antibody to bind. Make your purpose clear then we can discuss. ([email protected])
you can not use fluorescence Ab for IHC staining it should be Biotinylated or HRP conjugated. Fluorescence secondary Ab is proper for ICC or IF.
You can work with your primary abs. What you have to know is in which animal your ab is generated and weather your primary is a whole (H&L) serum because you need the c-fragment for binding your secondary ab. An example: mouse monoclonal primary ab FITC labeled ---> anti-Mouse labelled dirctly with another fluorphore or HRP or biotin ; rabbit polyclonal PE labelled primary ab ---> anti-rabbit labelled dirctly with Fluorphor or HRP or Biotin. If you use biotinylated secondary abs you should continue either with Strepavidin labelled with POD or Fluorphors or with Avidin-Biotin Complex (ABC) POD.
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