Full, complete and working antibody protocols are hard to come by and sometimes impossible to find. Customers would ask me if we have a protocol for an antibody, most times we did not. Therefore, for those who do not know what comes next, working up an antibody (antibody optimization) can be painful. I would first look at at the manufacturer data sheet for clues on where to begin. If that didn’t work, I would comb through dozens of journal articles looking for 1 piece of the protocol, a starting point. The starting point could be a tissue type(s), unmasking procedure like HIER at what pH, primary antibody dilution rate or anything that would give me insight into staining with an antibody for the first time. This optimization is a huge under taking that can take days or weeks, plus loads of reagents to figure out a working protocol.

If one of these antibody fits your needs, fortunately you will not have to go through that process. The first antibody targets human tissue only and can be stained with positive controls of Liver, Lung, Spleen, Testicle, Thyroid, and Tonsil. It is Abcam’s ab133616 EPR6855 rabbit monoclonal. I got this to work on formalin fixed paraffin embedded (FFPE) human tissues. The protocol is available here for purchase.

The Other CD4 protocol is for mouse tissue only. I don’t have it formally written up, now do I have have any pictures. This antibody is Ebiosciences cat# 14-9766-82, clone 4SM95 and a rat monoclonal. The protocol is for paraffin (FFPE) tissues used with HRP reagents. The positive control is mouse spleen. The HIER technique with Tris/EDTA pH 9.0 for 20 minutes works well. Using a primary antibody dilution of 1:100 for 60-90 minutes @ 60C and a rat secondary will help. The rest is up to you.

These procedures are not to be taken lightly, even though these protocols are on the easy side, they are still difficult. If you’ve never done IHC/IF or worked with antibodies, I suggest you run it with control tissues first before using it on any precious study tissues.