It’s Friday night and you could be out with your friends right now, but instead you’re tucked away in a dark little room filled with microscopes. Spending the evening in the lab seemed like a good choice at the time because you were certain this immunohistochemistry (IHC) was going to reveal some small—but important—mystery of the universe to you. But now you’re sitting here, cursing the universe and everyone in it, because all you see when you stare down into the scope is some indistinct fuzziness. Did the controls work?
There’s no sugarcoating it. It’s a fail.
IHC Troubleshooting
What’s next? If you’re like me, you’ll fling the slide into the trash with as much gusto as you can muster and head out to find your friends. Tomorrow, you’ll re-evaluate the experiment.
Where do you start? You probably know that a highly specific, high-affinity primary antibody is key to a successful IHC. But did you know that the companion reagents (i.e., buffers, etc.), which establish the pH and ionic strength of the system, are just as important? These reagents can influence the binding of the primary antibody to its epitope and dramatically affect the outcome of the assay.
To help you pick the best reagents for your assay (and make sure those Friday nights in the lab are worth the sacrifice) we will spend the next several posts reviewing how companion reagents affect IHC. As an example, we will describe our experience optimizing the protocol for one of our antibodies, PLK1 (208G4) Rabbit mAb #4513.
When we first tested this antibody, it failed to produce a signal with our standard protocols (Figure A). By changing the companion reagents one by one, we were able to generate a strong, clean signal without ever changing the dilution factor of the primary antibody.
Step 1: Antigen Retrieval
Cross-linking fixatives, like formalin, are often used to prepare samples for IHC because they preserve the structural integrity of the tissue, which helps maintain tissue architecture. Unfortunately, these fixatives may bury the epitope your antibody is designed to recognize.
Several methods exist for revealing epitopes that have been masked by fixation. These include proteolytic-induced antigen retrieval, which relies on an enzyme like proteinase K, or heat-induced epitope retrieval (HIER), which uses heat to break apart cross-linked bonds and unwind proteins. Either method can unmask epitopes, rendering them accessible to the primary antibody and amenable to staining by IHC.
At CST, our most common antigen retrieval method is HIER, so this is the method we will discuss in detail. HIER involves heating and then cooling the tissue sections while they are immersed in a solution with a defined buffering capacity. The pH of the buffer helps keep the proteins unwound after the temperature has returned to normal, so the pH range of the system should be optimized to the antibody-epitope interaction of interest. The slightly acidic buffer citrate (pH 6.0) is effective at unmasking a wide range of epitopes, but some epitopes may require a more basic buffer, like EDTA (pH 8.0).
We used citrate buffer for HIER while optimizing the PLK1 (208G4) Rabbit mAb protocol, because it works for the widest range of epitopes. We kept this method constant as we did a step-wise change of the other companion reagents.
Read the next post in this two-part series:
For more helpful hints, download A Guide to Successful IHC using the button below.
