We'll show you why antigen retrieval (AR) is important for immunohistochemistry (IHC), and give you tips on how to set up Heat Induced Epitope Retrieval in your microwave.
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Transcript:
- - What is antigen retrieval? And why is it so important for IHC? I'm Abbey Wells. - And I'm Franny Ambrose. - We are both research associates in the immunohistochemistry group at Cell Signaling Technology. - And this is CST Tech Tips.
Antigen retrieval, also known as AR, is one of the factors that can influence the strength of staining obtained in IHC. AR made be accomplished by treating the sample with either heat or enzymes. In this video, we'll focus on heat-induced epitope retrieval, or HIER for short.
Antigen retrieval takes place after deparaffinization of the sample and before the addition of antibodies to detect the target proteins. AR removes the cross-links formed as a result of aldehyde-based fixation and unwinds the protein's secondary and tertiary structure, making epitopes accessible to detection by antibodies. This can be particularly important for epitopes buried deep in the protein structure.
In your standard HIER protocol, samples are submerged in a buffer and boiled for a period of time, usually 10 to 20 minutes. Heating methods commonly used include a water bath, microwave, or pressure cooker. The level of retrieval can very among these methods, with water baths being the least effective and pressure cooker being the most effective. CST's IHC Protocol recommends using a microwave, which is effective for antigen retrieval, and you probably have one in your lab.
A common question we get is what unmasking buffer should I use for antigen retrieval. Many antibodies with work well with antigen retrieval performed with citrate buffer. Often with EDTA, you will observe increased signals, but possibly increased background as well. Antibodies that work with citrate buffer are likely to also work with EDTA, but may require further optimization in order to achieve the best signal-to-noise ratio. Conversely, if an antibody requires EDTA for retrieval, it is unlikely to work with with citrate. The antigen retrival buffer that we recommend with our products can be found on the product's data sheet.
Make your 1x citrate or EDTA and fill your container with 200 mL of that buffer. Fill any empty slots in your slide holders with blank slide. This will ensure that the buffer and heat distribution is consistent between experiments. Leave the lid slightly cracked to allow pressure to escape as this ensures the buffer does not boil over and expose the top of your slides. Also, overheating slides can cause a loss of buffer, which may result in tissues drying out. This can yield false negative results. A common misstep in antigen retrival is aggressive boiling. That can cause tissue to detach from the slide.
To optimize microwave time and power, we suggest performing a trial run. Use 24 blank slides and 250 mL of buffer and determine how long it takes to reach the initial boil. Next, determine the power level needed to maintain a sub-boiling temperature for 8-10 minutes. Once you have established the conditions for your microwave, perform the retrieval the same way each time, always using the same amount of buffer with empty slots occupied by blank slides.
You can find full application-specific protocols for each antibody on its product page at cellsignal.com.
If you have any questions about an antibody or a protocol, you can get in touch with one of our scientists at cellsignal.com/support. And 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. cellsignal.com/about
#CSTTechTips #Immunohistochemistry #IHC #AntigenRetrieval