Hi,

I guess you are dealing with FFPE material. Have you tried heat-induced antigenretrieval (HIER) with low or high pH?

Cheers,

Kristian Moller

For FFPE tissues, you can use any of the following heat retrival methods:

1- Heating with Tris buffer ( 0.1% trysin in PBS, pH 9.0) for 15 mins.

2-Heating in citrate buffer for 10 mins, low heat microwave.

NB. Make sure you use the pen blocker to demarcate the tissue borders before heating.

Let me know which one worked for you eventually.

Heating is one option, another option is to use some other pronase like Protease 14, Proteinase K or Trypsin. Using pepsin is trickey, need very specific pH, trypsinebis less sensitive. All needs 37C accapt Proteinase K which need room temperature. 

Dear Cora,

For FFPE you should as a rule include HIER (pH6, pH9) as well as a protease-step. The first choice would in my opinion be proteinase K which is use on a fairly routine basis to retrieve antigens for IHC. If this does not work would make a seires of lower diltuons together with the antigen retrieval steps. If this procedure does not give you the result you aim for, try another antibody.

When you have 10 um section, I am assuming frozen section. I have done Brdu 30 um IHC and I never do the antigen retrieval step, it is only for paraffin. I do the similar procedure to your acid treatment, 0.2 N HCl in 0.3 % triton X-100 and after this step did you neutralize with 0.1 M Borate buffer pH 8 15' at room temperature? May be this could solve your problem. Good luck