I did Immunofluorescence staining of DAPI using the lung tumor tissue frozen section which contains part of the normal site, but I find it hard to figure out why the DAPI staining (or other antibodies) intensity was weaker compared to the normal sites under the same condition. I repeated it more than once, I don't know what's wrong with it.
It's possible that the weaker staining on the tumor tissue it's because of greater tissue density and therefore less penetration of reagents to the tissue. Let's assume that the tumor portion of the tissue is more dense (for whatever reason, different ECM, secreted products, smaller cells, etc), and during fixation with paraformaldehyde a small difference in tissue density is exaggerated because this creates cross-links and thus can increase tissue density. When you carry out your detection, less reagent penetrate and signal is weaker. An alternative fixation method or a shorter paraformaldehyde fixation should circumvent the problem. Let us know if this makes sense.
Hi Dawn, with respect to the Dapi satin, it binds to dna such that the intensity is proportional to the DNA content. Differences in DNA can occur due to cell cycle (which is higl frequency in highly mitotic cells) and chromosomal instability, which occurs in the context of oncogenesis.
As for markers, many proteins change their expression level in cancer cells.
So what you are observing may not be artifact
Hi Dawn, like Oscar mentioned the tissue density of the tumor in comparison to the surrounding normal tissue may be significant enough to reduce antibody penetration, especially with the cross liking of paraformaldehyde. In addition to adjusting the fixation strength, you can also try a stronger detergent. I have to switch between Triton and Tween detergents myself to help optimize staining.
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