Experimental design: what controls should I include for Immunofluorescence (IF)? | CST Tech Tips

We'll explore three types of controls for your immunofluorescence (IF) experiment: positive controls, endogenous controls, and negative controls.
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👉Links mentioned in video:
🔗Cancer Cell Line Encyclopedia: portals.broadi...
🔗BioGPS: biogps.org
🔗Literature search: pubmed.org
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Transcript:
What controls should I set up for immunofluorescence experiments? I'm Ginny Bain, scientist in the immunofluorescence validation group at Cell Signaling Technology, and this is CST Tech Tips.
Immunofluorescence can be complex, and including controls can help your experiment be successful. Good controls can help you get your experiment back on track if something goes wrong, and they can also help you have confidence in your results. In this video, I'm going to talk about several types of controls, including positive controls, endogenous controls, and negative controls for immunofluorescence.
When starting a set of IF experiments, it's a good idea to include positive controls, which can give you an idea of what your protein of interest should look like in a cell type known to be positive for the target protein. Check resources like the Cancer Cell Line Encyclopedia, BioGPS or published literature to find known positives. Controls can also help you determine if your microscope is set up properly.
Related to these positive controls are single color controls, which are important to include when your experiment will have more than four fluorophores. Single color controls can tell you if one or more of your fluorophores is bleeding into a longer wavelength channel, which should be minimized. They are also used to set up spectral unmixing, if your imaging system has that capability.
Endogenous controls are used to assess the health of the cells or tissue in your sample. In addition, endogenous controls can tell you if something went wrong in the IF protocol, such as poor cell culture, or tissue preparation technique, improper fixation, or buffers going bad. You should be aware that if your cells are stressed, this can introduce uncontrolled experimental variables. If you suspect this may be the case, you can check by performing IF with indicators of DNA damage such as phospho-histone H2AX, or stain mitochondria to look for morphological changes indicative of damage. If working with tissue, you can look for signs of cell death with antibodies for cleaved caspase or PARP, or morphological changes such as condensed nuclei or cell blebbing. And if the tissue architecture has holes like Swiss cheese, it could be a sign of improper storage or embedding of frozen samples.
The third class of controls are negative controls. Performing a secondary-only control can give you an indication if your sample type is generally sticky, or contains cross-reactive immune cell types or Fc receptors, either of which will contribute to nonspecific background staining. PBS-only treatment can be used to assess autofluorescence in your sample, which could be from endogenous sources, or from sample processing steps, or a combination of both. Negative controls can be used to set the maximum exposure and laser power settings to avoid nonspecific signal, so you can be more confident the antibody is binding to its target protein.
Thanks for watching. You can find the application-specific protocols on the product page for each antibody at cellsignal.com. And if you have any questions about an antibody or a protocol, you can contact one of our scientists at cellsignal.com.... Good luck with your experiments!
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