Best Practices For Antibody Optimization In Immunohistochemistry?
"Typically, with a validated platform and detection system, there are only a couple of parameters to optimize for an IHC assay, such as epitope retrieval and antibody concentration," Nan explained. He pointed out that one can go and further optimize conditions such as antibody incubation time and temperature, or switch to a more sensitive detection system, but we would only do that in one of the following cases:
1) We run out of choices and only have a few available antibodies.
2) We want to maximize signal-to-noise ratio for a particular antibody.
Although it may look like a good idea to go with a comprehensive and large optimization matrix that includes all possible conditions (e.g. for epitope retrieval, there are acidic and basic retrieval, proteolytic retrieval, and sometimes no retrieval), but this might not be the best idea according to Nan. He described, "Well, granted that certain antibodies may work better with protease retrieval or no retrieval at all, experience has told us over and over again that a heated retrieval step almost always works better. Furthermore, the basic ER solution in the majority of cases give stronger staining comparing to acidic ER solution. Of course, acidic ER often yields cleaner background, but with a well-validated detection system, using basic ER provides higher signal-to-noise ratio with lower antibody usage and lower optimization cost for over 80% of antibodies. That’s why most of our customers choose to go with a smaller optimization matrix using basic ER as a start."
How To Design Titration Series As An Initial IHC Optimization?
The dilution of the primary antibody is dependent on the detection system one uses. Nan commented that if you're using one of the polymer systems, then you’d probably want to start with a lower concentration. Many recommendations from an antibody vendor’s website for IHC dilution may not be optimal for a polymer detection system. The concentration might be unnecessarily high. "Based on our experience, the starting concentration should not be higher than 5ug/ml for mouse antibodies or 2ug/ml for rabbit antibodies. If you need to go higher than that, then it’s not a good antibody, period. The results can be complicated by background staining." It is true, according to our experience from Boster's histology lab, that if you throw more than 10ug/ml antibody on to a tissue, you will get some staining, just don't trust those stains.
Troubleshooting IHC: What To Investigate When It Just Does Not Work
Also, When to Call It Quits and Move On to the Next Antibody
There are numerous troubleshooting guides online, and there are many factors one can try to optimize in IHC. Nan suggests to focus only on the few that matters. He mentioned, "If after a couple of rounds of optimization based on a well-designed parameter matrix as previously described, the staining is still poor, then the antibody is probably not suitable for IHC and extensive troubleshooting will not likely take you very far. I’d say the affinity and specificity are inherited properties of an antibody, so optimizing conditions will only push the signal-to-noise ratio to close to the best the antibody can do. Occasionally, altering incubation conditions can make a huge difference. For example, 4°C overnight incubation will typically give higher signal-to-noise ratio as it allows longer time to reach binding equilibrium while minimizing non-specific binding, comparing to RT incubation for 30min to 1hr. Unfortunately, it’s not really a practical workflow for diagnostic assays and almost impossible to automate. Therefore, for research purposes, it’s ok to try everything possible to push for the best results. However, for developing a more routine assay for large scale studies, especially clinically related studies, it’s better to try to find a different and more robust antibody."
The experiment itself sometimes is not to be blamed. According to Nan, the pre-analytical factors are just as important. "Samples that were prepared under sub-optimal conditions may generate very poor staining even when using a robust IHC assay," Nan said, "The major categories of pre-analytical factors include collection, fixation, processing, and storage. It is a whole different topic, but one needs to make sure that the samples are properly prepared before diving into tediously IHC optimization. Basically, garbage in, garbage out."
Another possible cause of poor staining is that maybe the assay just didn’t work on that day, and that can come from numerous reasons, such as reagents or instrument. If one suspects that the assay or instrument itself has some issues and decides to find the root cause, that could be another long journey. "But how do we know if a particular run is sub-optimal? The only way is to include control samples, which we can talk more in depth about another time." Nan said, re-emphasizing the importance of IHC controls.