The four most important types of controls you should be using if you want meaningful and comparable results.
It’s easy to get hung up on antibody selection, exciting multiplex ideas, and imaging your tissue sections when running immunohistochemistry (IHC). But without proper controls in place, it quickly becomes difficult, or even impossible, to distinguish false results from accurate ones. Also, if you don’t have proper controls, you can’t reliably compare results between experiments.
Here are our top four controls you need to think about before you start your IHC work.
1. No Primary Control
If you’re using indirect IHC, i.e., you’re using a primary and secondary antibody, this is a great way to make sure your secondary isn’t binding to non-specific parts of your sample. Of course, if you’re running direct IHC with a conjugated primary, this isn’t the control for you – take a look at the other controls here.
As the name suggests, for the no primary control you just run your IHC as normal, but you leave out the primary antibody. Any positive staining you see in this control assay means you’ve picked up a false positive and it’s probably time to change your secondary!
2. Blocking Peptide Control
The blocking peptide is the antigen we use for immunization during development of the primary antibody. As such, this peptide should absolutely bind (or ‘block’ – hence the name) your primary antibody. For IHC, the blocking peptide control is a great starting place to get more insight into antibody specificity. This control is also often called a pre-absorption control.
In this control, you incubate your primary with the blocking peptide before applying it to your tissue. You then carry on as normal. Any staining seen in this negative control is likely because your antibody is binding off-targets.
You can read more about blocking peptides and how they work on our blocking peptide page.
3. Positive and Negative Tissue Controls
Positive tissue controls use tissue types that you know express your protein of interest. If you’re certain your antibody is working as intended and you get positive results here, then you can conclude that your assay is also working as it should. One less thing to worry about! But, if you get no results here, then you might want to have a look at our troubleshooting guides as something is awry.
Negative tissue controls on the other hand are, as you’ve probably already guess, tissues that do not express your protein of interest. These types of controls highlight false positives, which are very likely due to non-specific binding on the part of your primary antibody.
Negative tissue controls in IHC are often hard to come by (but thankfully are becoming more common!) as they usually require genetic modification beforehand by something like knockdown (KD) or knockout (KO).
If you don’t have access to control tissues and need to know how expression should look in a particular time, you definitely go look at The Protein Atlas. You can find loads of IHC results in lots of different tissue types.
4. Endogenous Background or Autofluorescence Control
Plenty of tissue types will naturally produce some degree of background staining or autofluorescence. Things like collagen and elastin are major culprits but so are eosinophils, lipofuscin, and certain types of embryonic tissues like yolk.
Sadly there’s no one-size-fits-all solution for this. You can start by imaging your tissue without any staining to get an idea of how much background your tissue naturally generates. You could also try using a fixative with 1:1 acetone:methanol, which can reduce levels of autofluorescence in some cases. Certain dyes, like Sudan Black, can also be effective but these can also negatively affect the signal.
The truth of the matter is that you’re going to have to either get a handle on the background before your stain and adjust for this spectrally or try several other steps specific for your tissue. Sorry!
If you’d like to read a little more about IHC controls and standardization from a more academic viewpoint, there’s a nice editorial from a good few years ago now by Torlakovic et al., which is really aimed at diagnostic IHC. There’s also a very interesting Nature article from 2016 by Uhlen et al., where they lay out their five pillars of antibody validation you’ve probably heard about. These pillars have been discussed and expanded or simplified in a host of other more recent papers. If you’re looking for IHC protocols, we have a very detailed step-by-step IHC guide for indirect and direct methods.