I am interested in detecting a protein that upon activation translocate from the cytoplasm to the nucleus. I want to detect this protein in the nucleus using IHC and mouse tissue. How can I introduce the antibody to the nucleus of cells in tissues? Any ideas
Hello,
Step 01: It is necessary to start with the development of a protocol of unmasking (acidic or basic unmasking solution, usgae of bain marie, microwave ...) especially if your protein will be associated with the DNA of the cell. This step involves making your proteins accessible by antibodies. Step 02: Perform an ordinary immunohosto- chemistry. However, you obviously start directly with an IHC and if it does not give results you can get back to unmasking. Good luck
Hi,
In IHC it does not matter in which compartment the protein to be detected is situated. Since you make sections, proteins in all compartments are accessible.
In the datasheet of your antibody you can find information about anitgen retrieval (unmasking). If you use paraffin embedded tissue, check whether your anitbody is suitable.
Thank you Rafic and Ronald....
Rafik, I think your way of unmasking is appropriate. Do you know any papers that does this kind of protocols? I want to start searching for this but not sure where to start!
Ronald, I am not sure that the antibody can enter into the nucleus unless I did something to open the nucleus in my tissue! This is challenging to achieve in tissues may be using cells is a better choice!
Hi,
The protocol that I have was effective for the detection of steroid receptors (receptor of androgens and estrogen receptors (ESR1, ESR2). This allowed me to locate my receptors at the nuclear as well as cytoplasmic level.This same protocol Has given excellent results for the detection of GPR30, a receptor coupled to the trimeric G proteins which can have a cytoplasmic, nuclear and membrane localization.You will find my articles detailing these protocols.
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