Let's explore the difference between fixatives: crosslinking formaldehyde versus methanol and other alcohols, for immunofluorescence (IF).
👉Subscribe: kzfaq.info...
👉CST Protocols and Troubleshooting: cellsignal.com/protocols
👉Get in touch with a CST scientist: cellsignal.com/support
💡 Get insights and advice on techniques, publishing, and navigating a scientific career: www.cellmentor.com/
Transcript:
What's the difference between crosslinking aldehyde fixatives and alcohol based fixatives for immunofluorescence? Hi, I'm John Burford, senior research associate on the Immunofluorescence Validation team here at Cell Signaling Technology, and this is CST Tech Tips.
Fixation is an important step in the IF protocol. Fixation works by stopping the activity of endogenous proteases in the sample so that proteins aren't lost, and by preserving the sample architecture so you can then introduce antibodies to detect your protein of interest.
Ideally, fixation would not alter the ability of antibodies to detect proteins, but unfortunately fixatives do have an impact on the epitopes, and can also affect the fluorescent properties of the sample itself. Because of this, it's important for you as an antibody user to understand the different types of fixation approaches.
For IF, there are two main classes of fixatives. First, are aldehyde based crosslinking fixatives which form chemical bridges between the lysine groups of proteins, providing structural stabilization to the cell. There are three potential challenges with crosslinking fixatives. One, when proteins are packed tightly together and highly crosslinked, it may be difficult for the antibody to access the protein of interest. Second, if the antibody does make it to the protein, the epitope it recognizes may be chemically altered, effectively inhibiting antibody binding. Third, aldehyde fixation can create autofluorescent byproducts in the sample that increase nonspecific background in IF. This can be somewhat avoided by choosing longer wavelength, lower energy fluorophores.
The second type of fixation approach is alcohol based organic solvent fixation. This works by stripping the proteins of their hydration barrier, effectively dehydrating the sample. This washes away soluble proteins, but precipitates the proteins that remain and alters their tertiary structure. This can help antibodies access otherwise hidden epitopes, at the cost of losing soluble proteins.
Here at CST, we test all IF antibodies so you can be confident they'll work with the recommended fixation protocol. When designing your IF experiments, check the product pages at cellsignal.com for the appropriate protocol for your antibody. Any time you have questions about an antibody or a protocol, you can reach out to one of our scientists, such as myself, at cellsignal.com/support.
For more tech tips videos, subscribe to our channel, and we'll see you next time. Good luck with your experiments, thank you.
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. cellsignal.com/about
#CSTTechTips #Immunofluorescence