IHC Troubleshooting

Troubleshooting IHC

In IHC there are many intricate steps to perform. The process is delicate, so problems will eventually appear. You need to know what to do in that event. Here is a quick, handy guide that can help you diagnose and solve different IHC issues.

Weak Signal

Getting a poor staining result is the most common problem you are likely to run into in IHC. Unfortunately, there are many reasons this issue will appear. Your tissue may have not been taken out of the paraffin properly before mounting. You should always be sure to use fresh xylene for the removal of paraffin. Also, consider using longer incubation times.

You may also find that primary antibodies are the problem. Antibodies are less stable after they have been diluted, so long-term storage may break them down. The antibody may also be too dilute. The solution in each case is to prepare a fresh batch of antibody for use. Make sure you aliquot frozen antibodies in order to prevent repeated freeze-thaw cycles.

Fixation is another area of the protocol that causes headaches. Too much is bad. Not enough is bad. If you suspect the tissue is fixed too much, then the antigen is probably masked by the excessive crosslinking. Consider using an antigen retrieval method to circumvent this problem.

You may also find that the secondary antibody is the problem. Ask yourself the obvious questions. Did you dilute the antibody too much? Is the secondary antibody compatible for detection of your primary? You may also find that the detection reagents you are using are incompatible with the enzyme conjugated to the secondary antibody. Make sure you understand the specific procedure you're using in great detail.

Too Much Signal

Usually a troublesome strong signal is caused by the antibody concentrations being too high. In this case, run a set of experiments that use different concentrations to determine the optimal detection conditions.

A less obvious issue with your samples could be drying. Never let your tissue dry out during the staining procedure.

Too Much Background Staining

High background signal is a rather annoying result. It means you will not be able to interpret the staining very easily, and it certainly is not publishable. Typically, this problem occurs from inadequate washing. Consider increasing the number of wash steps in your procedure.

The detection system you use will also affect the amount of background you see. Tissues with high natural peroxidase activity, for example, will be unsuitable for detection by horseradish peroxidase. The addition of the diaminobenzidine reagent will result in high background. Tissues that contain natural peroxidase, alkaline phosphatase, or biotin will need to be handled carefully.


Antibodies can also cause high background signals. The primary might be too high in concentration. The secondary may cross-react with proteins in the tissue if it comes from a common source. For example, if you are studying rabbit muscle, you probably do not want to use an anti-rabbit secondary antibody.
For more information about IHC troubleshooting please visit http://http://www.genetex.com/Web/Troubleshooting/TroubleshootingList.aspx. 

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