There are several steps in the IHC protocol that can impact your results. In this video, we’re going to focus on antibody diluent, which is the solution that primary antibody is diluted into for incubation with a tissue sample. It’s important to use an antibody diluent that works with your particular antibody, as the choice of diluent can impact the standing results, sometimes significantly.
The optimal antibody diluent supports antibody epitope interactions and specificity, and ultimately, the strength of your staining. Differences in diluent pH and ionic strength can increase or decrease specific interactions of the antibody with its target. Additionally, diluent properties may affect non-specific interactions with off-target molecules and can affect antibody stability. These problems can be overcome by addition of serum proteins as stabilizing agents, along with optimizing the pH. If that sounds like a lot of work for you to optimize your protocol, the good news is that CST recommends the diluent that should be used with each antibody.
Sometimes customers ask us if they can use Signal Stain diluent with all CST antibodies. SignalStain antibody diluent does work well with many of the IHC validated antibodies in our catalog, but it is not compatible with every one. For some CST IHC approved products, stronger signal may be achieved with an alternate diluent such as TBS-T +5% normal goat serum.
Customers also ask what could happen if they use a diluent other than the recommended one. Certain CST antibodies may yield nearly identical results in a wide range of primary antibody diluents. We have found, however, that some antibodies are more sensitive to the choice of diluent than others. This is one of the steps to keep in mind when troubleshooting low signal intensity.