Learn how choice of antibody diluent can affect staining results in immunohistochemistry (IHC).
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Transcript:
- - What diluent should I use for primary antibody incubation in immunohistochemistry? I'm Chris Grange from the IHC group at Cell Signaling Technology and this is CST Tech Tips.
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 staining 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 SignalStain 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. You can find the recommended diluent for your antibody in the IHC protocol section of the antibodies product page on [ Ссылка ].
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.
Thanks for watching. You can find full protocols, including recommended diluents for IHC on the antibody product pages at cellsignal.com. If you have further questions about an antibody or a protocol, you can reach out to one of our scientists at [ Ссылка ].
For more Tech Tips videos, subscribe to our channel, and we'll see you next time. Good luck with your experiments.
👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. [ Ссылка ]
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