When setting up IHC experiments, cell pellet controls can tell you whether the protocol is working or needs to be adjusted.
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Transcript:
How do I use cell pellet controls in immunohistochemistry? I’m Chris Grange from the IHC group at Cell Signaling Technology, and this is CST Tech tips.
It’s easy to apply an antibody onto an IHC sample and obtain a signal. But proper controls are needed to give you confidence that your antibody is detecting its intended target, and not something else.
Keep in mind that controls that are used in another application, like western blot, only provide information about antibody specificity in that setting. So when you’re setting up your IHC experiments, you’ll want to incorporate appropriate controls for IHC.
Today, I’m going to talk about one type of IHC control - cell pellets.
Formalin fixed, paraffin-embedded cells can be easily obtained, and are convenient as both positive and negative controls. Cell pellet controls are often used when starting a new set of experiments or receiving a new antibody, and you want to know that it will perform as expected with your IHC protocol.
Tissue-based controls can be useful - if there is a tissue known to express your protein highly, and another known to have much more restricted expression. But for many proteins, it’s not so straightforward to determine which tissues to use as controls. In this situation, cell pellets with differential expression of your protein can be valuable.
Ideally, you’d have one cell line that endogenously expresses the target protein as the positive, and another that lacks expression as a negative, to provide a representative picture of target expression levels. If these are not available, cell lines that express the protein at higher versus lower levels can often be informative.
Alternatively, you can use cultured cells transfected with the protein as a positive control, and mock transfected cells as a negative.
If you are using positive and negative cell lines and you see staining in the negative cell line, then protocol adjustments are needed. If with protocol adjustments or antibody titration you are unable to eliminate the signal in the negative cell line, then the antibody you are using is not likely to be suitable for IHC.
Now, let’s talk about treatments, which can be used with cell pellets and sometimes with other types of controls. The expression or activation state of some proteins can be modified by treating the cells with ligands such as growth factors or drugs. For phospho-specific antibodies, this entails treatment of the sample with phosphatase to confirm specificity. If available for the target of interest, these systems can be some of the most informative cell pellet models.
If the cell pellets give clear results, you can have some confidence that the antibody and protocol are compatible. This is important, but it bears mentioning that this cell pellets alone are not sufficient to confirm antibody specificity. Additional controls are needed, but the cell pellet controls are a great start.
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