We have raised a rabbit antiserum against a protein coupled peptide. This peptide has been reported to be antigenic and some papers have been published with a commercial preparation raised in the same way. Our antibody has been purified by affinity chromatography with the antigenic peptide and works well in Western blots, but we do not see any staining in FFPE-tissue sections, not even in overexpressing cells. We normally use an automated staining system but have also tried to do it by hand with a normal secondary HRP conjugated antibody. There is absolutely no staining, not even "unspecific". Is there anything we can do about it?
Dear Norbert,
in my opinion there could be two possibilities what might went wrong.
1) Primary antibody did not bind to the epitope since it might be masked. Have you used heat-induced epitope retrieval (HIER)?
2) The secondary antibody does not recognise the binding site of the primary antibody. In this case you could try to use a different secondary antibody or use a kit to label your primary antibody with HRP or a fluorochrom.
Best
Lukas
Epitope unmasking in citrate buffer under heat in a microwave oven or a pressure boiler (HIER), as Lukas has suggested. I would try that as a first step, and it will probably work
Assuming there is no major technical issue (antigen retrieval, secondary system, controls are fine etc.) then there are still several factors that can prevent a positive staining. It could be that the epitope cannot be recognized on FFPE sections. It is not uncommon that an antibody works fine on Westerns but not on FFPE tissues. If you have the option, you could try frozen sections. There is still the option to try different antigen retrieval buffers and protocols or use a much more sensitive detection method, e.g tyramides.
Have you directly compared the tissue material in Western and IHC or only confirmed that the antigen can be detected when overexpressed in vitro? Using frozen tissue, it is relatively easy to perform such a direct comparison and rule out that the antigen is really expressed in vivo (I assume you did something like inject transfected cells in mice and harvested tissues samples??). If you have not done this type of comparison then it could be that the antigen is not that much overexpressed and therefore you can't see it (low likelihood but possible).
If you don't have frozen material, you still can run a Western by using e.g.a kit from Qiagen or prepare the required reagents yourself. You can get lots of decent protein out of FFPE sections using heat and an appropriate extraction buffer.
If you already have shown by Western that the FFPE tissue expresses the antigen, I would consider frozen tissue for staining. Frozen can have disadvantages and not all antibodies work on frozen sections. I think it often simply the lack of good fixation and several proteins are just "washed out" from the sections before they can be fixed properly.
Hi Norbert. I agree with above comments. If the objective is to test the antibody I would also try to tested it using other known positive tissues to be used as positive control, as well as compare your antibody with any comercial one if available. Test various Ab dilutions and different secondary Ab. The thickness of the section , how the tissue was processed also influence the results. All steps from tissues collection, preparation of the fixative etc, could be revised. here is a publication that may help
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