Like to know other researchers opinion on quantifying some immunohistochemical/immunofluorescence data, I double labeled some markers using IF on FFPE tissue samples, I also have the adjacent slides of the IHC (same marker), should I count the co-localization using IF (or only use this as proof of existence of the co-localization) or should I look through the adjacent IHC slides.
Fluorescence is much more sensitive than whatever system you may use in bright field. As you are analyzing, you can also multiplex many antigens and now with the OPAL system you can arrive up to 10. However, in fluorescence everything is amplified, and in case you have differences in background, quantification of your antigen can be compromised (if you do not have the right hardware and software).
This is true if you are comparing different slides or different histospots in a TMA.
However, what you are suggesting should be free from such inconvenience. Co-localization and differences between adjacent tissues should be easily quantifiable even with free software such as Cell Profiler, since background will be the same.
Hope it helps.
I agree. Immunofluorescence is better to indicate co-localization of markers. IHC could be confusing because marks can overlap and won´t be discriminated as easily as two different fluorochromes.
If you are more familiar w/ IF procedures, and there are no background issues use those slides. The IHC DS procedures are usually a little more tricky. I'll refer you to the book: Immunoenzyme Multiple Staining Methods, CM van der Loos, BIOS Scientific Publishers Limited/Springer, 1999. I prefer IHC methods to IF because you have the added benefit of the tissue structure in LM, but if it is just quantification ,IF should be fine.
We routinely use immunofluorescence microscopy (IFM) for looking at cell marker expression in situ, as well as for co-localization analysis. If you also need to visualize the tissue architecture, actin-binding phalloidin conjugated to a flourophore is a great choice. Using phalloidin-Alexa 350 ("blue" channel), you can stain for 3 other cell markers (FITC, PE and APC channels). Specificity of antibodies is very important for IFM. Often cells co-express various markers, up-regulate or down-regulate marker expression, thus all these factors need to be taken into consideration when doing this kind of work. There are great softwares available for quantification and co-localization analysis. We use Volocity and are very happy with it. If you stain parallel sections for IHS, you can also verify the data that you acquire using IFM.
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