Biology Forum Molecular Biology Difference between ICC and IF

last updated by simina 11 years ago
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    • #10110

      What is the difference between immunocytochemistry and immunoflourescence?

      As far as I can tell, it is basically the same principle with the primary and secondary antobodies. Is the difference in what you are actually staining? ICC stains what exactly? The cell membrane only? Inside of the cell also?

      Is ICC just a type of IF?

      Thanks for your help in clearing this up for me!

    • #85946

      The basic approach of using antibodies to mark structures is the same. However, the way to determine the location of the antibodies differs.

      Immunofluorescent methods use antibodies labeled with fluorescent moieties (fluorochromes). Usually these are conjugated with a secondary antibody which is used to bind to a primary antibody bound to cellular antigen. The antigen can be outside the cell or inside, depending on how the cell is fixed (some fixation processes made cells permeable). The fluorochrome is detected by fluorescence microscopy, always involving excitation of fluoresence with a defined wavelength band of light and detection of the longer-wave emission from the fluorochrome. Generally for epifluorescence microscopy, light from a UV-vis light source is passed through an excitation filter and a dichroic mirror, then the fluorescence emitted from the sample is passed back to the dichroic mirror and through an emission filter to the ocular lens or camera.

      Immunohistochemical methods encompass a broad range of chemical reactions used to produce a microscopically visible signal, usually by producing a stain or sometimes emitting photons or producing a fluorochrome. A few examples are using secondary antibodies conjugated with horseradish peroxidase or with beta-galactosidase, each of which can catalyze reactions producing stains and the stains detected by microscopy. Lumogenic substrates can emit photons when they undergo reactions. Reactions producing fluorochromes can be images using epifluorescence microscopy.

      I hope someone else will chime in — this really isn’t my field, but I’ve worked a bit with fluorescence.

    • #85947

      I’m no expert, either. I guess the two terms can overlap, but neither is completely covered by the other. Immunocytochemistry is the use of immunological techniques to study celluar constituents. When the “immunological method” also involves fluorescent labelling, then the two terms would be referring to the same thing in that instance. Immunofluorescence is a general term for using antibodies tagged with a fluorescent label. Whenever such an antibody is used to study cellular constituents, then the method could be described as both immunocytochemical and immunofluorescent. But I can imagine situations where an ICC method is not fluorescent (say the antibody is tagged with peroxidase rather than a flourescent probe). I have a harder time thinking of an IF experiment that is not also ICC, but I bet it’s possible. Practically speaking, I think it closer to the truth to say that IF is a subset of ICC than the other way around, but I also don’t think the two terms necessarily restrict each other.

    • #113591

      I also have some doubts about fluorescence microscopy!? What technique we can use to visualise by fluorescence microscopy a transfected proteins that laks any fluorochrome in mammalian cell cultures? Does anyone have any ideas?

    • #113594

      Typically you would use an antibody to bind to the protein of interest and then a secondary antibody carrying a fluorescent tag to detect the first antibody.

      The two-antibody approach is cheaper than making a custom-labelled fluorescent antibody for every antigen you need to detect. For instance, the first antibody (called the primary antibody) might be raised in a rabbit, while the second antibody (the secondary antibody) is a generic anti-rabbit IgG antibody carrying a fluorescent tag. Once someone has made a fluorescent antibody to rabbit IgG, it can be used as a secondary antibody along with any rabbit IgG antibody as a primary.

    • #113614

      Is this technique has a special name, for example FRET or you would suggest to give this explanation straight away, without mentioning any name? It is FRET method quite similar to this one? Thank you!1

    • #113615

      This is how immunohistochemistry is typically done, so that is probably enough of a name. However, you could specify the antibodies, for example: immmunihistochemistry using goat anti-VEGFR primary antibody and anti-goat-IgG secondary antibody conjugated to horseradish peroxidase.

    • #113616

      great! Thank you very much for your help

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