I have attempted to detect a nuclear protein (RUNX2) and images by the supplier of the antibody shows the protein being present within the nucleus of the cell. I have used the primary in a 1:100 dilution and the secondary (Alexa 488) in a 1: 400 dilution.
(https://www.thermofisher.com/antibody/product/RUNX2-Antibody-clone-6HCLC-Oligoclonal/711519?selectedApp=IF)
Are there any specific steps that requires permeabilsation of the nuclear envelope in order to successfully deliver the antibody to the nucleus? This is a concern since I see unspecific binding of the antibody within the cell body, but certainly not in the nucleus of the cell. Any advice would be appreciated.
Thanks in advance.
If you are using standard immunohistochemistry protocol, than there are not any specific steps, everything is covered for nuclear or non-nuclear.
What kind of sections you are using, paraffin-embedded sections (antigen retrieval will be needed), if frozen sections or cell monolayers (antigen retrieval generally not needed). Best thing will be to call the company technical support where you bought antibody. They will resolve your specific issue very easily since they have tested antibody before started selling. Best wishes
You can find the explanation here.
https://blog.citeab.com/saponin-tween-20-triton-x/
Hope that helps
Best.
Runx2 isn't necessarily nuclear. It depends on its state, and when active, it's mostly nuclear. You can do a nuclear:cytoplasmic ratio as a measure of its activity in your different treatment groups.
As long as you permeabilise with something, i.e. 0.1-0.5% Triton X-100 or something similar, then you should be fine.
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