Hi,
I am trying to perform an IHC targetting a protein tagged with an Histidine tail. For this reason we ordered a recombinant Anti-6X His tag® antibody, which brings the following dilution recommedations depending on the application (image).
As you can see, the IHC dilution recommended is for paraffin-embedded samples and my samples are frozen and embedded in OCT( Optimal cutting temperature compound).
I will try to make different dilutions in parallel to optimize it, but, should I use dilutions near the IHC-P recommendation (1/16000) or to the one of ICC (1/100)?
*I thought It could be more similar to the immunocytochemistry because my samples have been directly frozen and the target protein should be more exposed than in tissues treated with paraffin, but I would like to listen to other opinions.
Thank you for the advice in advance.
The 16000 actually looks like a mistake to me, I've never seen such a low dilution recommendation for histology anywhere. Best check with the technical support of the supplier, they are usually quite helpful.
If not, I would try something like 100, 300, 500, 1000, 2000. Lower dilutions than that are often not practical (if you don't want to stain 100 samples at once).
If the company did not checked IHC with frozen section, you can try to see from the references they have for the antibody for frozen section IHC. But regardless, you are right, ICC is more closer than paraffin IHC dilution factor for frozen section IHC immunofluorescence. Also, I think you may have to check some dilutions to get optimize if you don’t see good results with 1:100 DF. May be at 1:200, 1:500, or 1:1000.
HIS tag produces a very strong signal. It is potentially further enhanced by unmasking when you boil paraffin slides in citrate or similar solution, even thought the HIS tag is either on C or n-terminus. With DAB detection, and 3-step antibody detection (primary -2ndary biotin -and avidin-HRP) there is substantial signal amplification, the dilution may actually be 1:5000 -1:10,000. When you do ICC, you do primary and then secondary-fluorophore, and no unmasking. Testing several dilutions of course is the best approach but i do recommend testing one at 1:5000 to 1:10,000 if you are using DAB as a substrate (more sensitive then fluorescence) and VectorLab or similar 3-step detection approach. Dont forget to quench endogenous peroxidase if you do HRP-DAB detection. Good luck
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