Dear colleagues!

Please help me to solve a strange problem that I met trying to detect p53 with indirect immunofluorescence.

In brief:
Primary AB - SantaCruz rabbit polyclonal (FL-393), works well on immunobloting, good for WB, IP, and IF as the manufacturer says.
Secondary AB - SantaCruz goat anti-rabbit FITC. The antibody is fresh, gives a good green emission when a drop of the AB is placed on a UV transilluminator or under a UV lamp of fluorescent microscope.

Cells - HEK293. The cell line producing p53 in an industrial scale. The p53 (huge amount) is located exlusively in the nucleus which was confirmed with immunoblotting of citozolic and nuclear extracts (well it is known since 100 years ago or so).

When I try to perform the IF I do not see nothing that I could call a specific signal, just SMALL more or less bright green SPOTS at the location that might be somewhere in the nucleus.

Frankly speaking, I do not know if it is a specific property of the HEK293 cells, but what I expect (well, many people would expect the same) is a more or less uniformly stained nucleus.

Looks like p53 occupy a specific domain in the nucleus (smile) or I get something absolutely wrong. I could say also that it looks like p53 "leaks" from the nucleus. Is it possible after fixing? - I ask myself.

What I tried with different dilutions of antibodies (I started with 1:400, now I use 1:100-1:50):

1. 2% paraformaldehyde - 0.2% Triton-X100 - blocking solution (5% newborn calf serum in PBS) - 1st AB in the blocking solution - wash with the blocking solution - 2nd AB in the blocking solution - wash in the blocking solution - wash in PBS - mounting

2. The same as 1st with 0.5% Triton X-100

3. The same as the 2nd with 0.2% NP-40 in the blocking solution

4. The same as the 1st, 2nd, and 3rd with fixing in ice cold methanol

5. The same as the 4th with ice cold acetone after fixing

6. The same as the 1st with 1.5% BSA in the blocking solution

7. The extreme one - the same as the 2nd with NO serum or BSA in the blocking solution, just PBS.

I ALWAYS SEE THE SAME SMALL SPOTS (help!).

In parallele, I set up the same IF with other pair of ABs - goat Lamin B/mouse anti-goat Texas Red (both from SantaCruz), everything works properly, I get the expected "rings" of the nuclear envelop.

There is no problem with filter sets, I see drops of Texas Red or FITC with appropriate filters of the microscope.

So, my question is: what else I should try to see what I want?

"Use other antibodies" would be the most dramatic answer, but it is still accepted (if there is no other ideas).

I would greately appreciate any comments.

Thank you in advance.

Alexei.

Have you checked the literature where they show the immune staining of p53? I am sure this must have been shown before. Try to see the difference between your method and the published one. If still no change, I guess you have to change the primary antibody.