Troubleshooting Immunofluorescence: Excess (Bright) Signal | CST Tech Tips

Does your immunofluorescence (IF) look like a bright mess? Let's troubleshoot some potential causes.
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Transcript:
How do I troubleshoot excess signal in immunofluorescence? I’m Ginny Bain, Scientist in the Immunofluorescence validation group at Cell Signaling Technology, and this is CST Tech Tips.
When you’re analyzing data at the microscope, what you see matters. When you see what you expect to see, that’s wonderful. But here’s what to do when you see something unexpected. In this video we’ll talk about what too much signal means, potential causes, and solutions for this problem.
First, too much signal can be caused by overfixation. In this case your entire specimen will have a glowy appearance. To address this, try a shorter fixation period. Fixation time depends on the size of the tissue sample, but for small pieces of tissue and cells, 15 minutes is usually sufficient. Make sure to use 4% formaldehyde at room temperature or 4 degrees celsius.
Another common problem we see that causes too much signal is when the cells or tissue have dried out after addition of protein-containing buffers. This causes intense autofluorescence. To prevent this, make sure to change your buffers quickly and use sufficient buffer to prevent drying during incubations. You can also make a humidified chamber, look for that video coming later.
If your negative control slide looks clean but you’re seeing too much signal in your experimental samples, you may have used too much antibody. It is possible to get too much signal when excess antibody is used, so make sure to titrate your antibodies appropriately or use the recommended dilution found on the product page.
You may see excess signal due to secondary. Any secondary conjugates that are present will fluoresce even when not bound to primary antibody. If the entire field of view on the microscope is glowing, then you may not have washed the secondary off. You can fix this with extra PBS washes.
It is also possible to get nonspecific binding of the secondary to your sample when you incubate for a long time or at high temperature.
Make sure not to leave secondary on your sample for more than 2 hours at room temperature to prevent this.
Finally, if the signal is specific, but really strong, it can saturate the detector, losing dynamic range, and may also cause spectral bleedthrough to another channel. If this happens you can try using less antibody,or simply adjust the exposure time or laser power to take advantage of the full dynamic range.
I hope this video is helpful! Please subscribe to our channel for more Tech Tips and IF troubleshooting videos.
You can always find application specific protocols for each antibody at cellsignal.com. And if you have questions about an antibody
or a protocol, contact a scientist at cellsignal.com/support.
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
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[@sarikayadav5882]

hello there! I have a question about what should be the best fixative for intranuclear proteins.

[@cellsignaldotcom]

Hi Sarika. If you are using a CST antibody, you can simply type in the product number in the search bar on www.cellsignal.com. Scroll down below the images and click on the button for "Protocols". If that product has been validated for immunofluorescence you can select it from the drop-down menu which will bring up the IF protocol and the fixative that we used in our validation process. (If you are performing ChIP, IHC, or another assay just select the relevant application.)
If you still require technical support, the best way to have your question routed to the scientist who can help is to fill out the form on cellsignal.com/support.
In the meantime, if you missed our Tech Tip video on fixatives, you can watch it here: th-cam.com/video/NzeAYvQg8EI/w-d-xo.html