Looking to perform IHC co-staining with CD3 (cytoplasmic) and MUM1 (nuclear) antibodies.
CD3 polyclonal rabbit, HIER Na citrate pH6.
MUM1 is monoclonal mouse, HIER EDTA pH9.
I've tried HIER on both antibodies using same retrieval method but they are not compatible.
I was thinking of performing the first stain uses pH6 CD3 then do a second retrieval at pH9 MUM1 and vice versa.
I have DAB and Magenta chromogen to use.
Does anyone know is DAB would be damaged during a second round of HIER?
DAB is heat resistant. It should not be damaged by the heat of a second HIER step. See attached chromagen substrate chart from Vector Labs. DAB is heat resistant for peroxidase detection. There are a few others for Alkaline Phosphatase based detection methods.
The question remains whether your antigen will be retrievable after going through the first HIER treatment. Unfortunately this is antibody-specific and will need to be tested empiracally. Make sure to use controls for all scenarios (primary 1 only, primary 2 only, primary 2 only with both HIER steps, etc) to narrow down any issues.
Renee R Donahue thanks for taking the time to respond. I had planned to test the effects of a second HIER on both Magenta and DAB. These was a slight communications error and these slides ended up getting counter stained and mounted rather then left aside.
The good news is that I had other variations running the same day and results suited an different approach.
I performed HIER using EDTA pH9 and and altered the dilutions of CD3 to remove previous background but still maintain the required staining.
Using the Magenta first to stain for MUM1 followed by DAB for CD3 the result was beautiful contrasting staining which visualised both stains perfectly.
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