Optimization of IHC Experiments

Optimization of IHC

Immunohistochemistry (IHC) is an extremely valuable technique used in a variety of different settings to determine protein location and amount. It is also a rather challenging technique that requires troubleshooting and a good understanding of principles. Here are some commonly-encountered concepts in IHC.

No or Low Detection of Protein

Sometimes proteins adopt a different conformation from their native state after fixation. This can cause the part that is detected by the antibody to become hidden. Proteolytic enzymes like trypsin can help unmask this portion of the protein of interest and allow it to be detected once more.

Non-specific Staining

Two systems are commonly employed to detect where antibodies have bound in the cell. They are horseradish peroxidase and alkaline phosphatase. Detection by these enzymes relies on the reaction of a substrate. Unfortunately, there are many different proteins that occur naturally in the cell that may be able to catalyze the same reactions. This will result in non-specific staining of your tissue. The solution to this problem is to pre-treat with a solution that will destroy the activity of these enzymes. Hydrogen peroxide can be used to inhibit endogenous horseradish peroxidase, and weak acid solutions will permanently disable alkaline phosphatase.
Another cause of non-specific staining is non-specific binding of the antibody to other proteins. This can be averted by applying a blocking solution to your tissue sample before adding the primary antibody. There are many common blocking reagents in use today, but they will all compete with the antibody for the non-specific binding sites available in the sample. This will help ensure that the antibody is only able to bind its target.

Dilutions

Antibody detection is entirely structure-based. The protein of interest needs to have the right structure, and the antibody needs to be properly folded to work. Antibody structure is heavily dependent on its environment. This includes the concentration of surrounding antibody molecules. Dilution can present a situation where the antibody's structure changes slightly and causes it to lose the ability to detect a protein specifically. In this case, you lose signal or gain too much non-specific staining. There is no simple solution for this issue, but you can take several things into consideration when preparing a dilution.
First, be careful with the properties of your buffer. Make sure it has a pH around 7.3 or 7.4 to assure proper ionic interactions. Large changes in pH will alter the charge of antibodies and cause deformation of their structures. Second, be careful with how much preservative goes into the dilution buffer. Sodium chloride and sodium azide are good at preserving solutions from microbes, but they can affect the structure of antibodies when they are at high concentrations. Third, try to avoid diluting too much. In general, antibodies are more stable when they are concentrated, so don't use a very dilute antibody solution very many times. Fourth, keep in mind that the common buffer Tris is very sensitive to temperature in terms of pH changes. If you set the pH at room temperature, you may find that the antibody structure is lost after putting it in the refrigerator.

Wash Buffers

There are two commonly-employed wash buffers used in IHC. These include tris-buffered saline and phosphate-buffered saline. Wash buffers are used to remove the non-specifically bound antibodies from the solution to arrive at a clean picture. When preparing a wash buffer, use fresh reagents and sterile water. Do not mix different types of buffers, and always carefully date your bottles before use.

Improvement of Chromogenic Signal

Diaminobinzidine (DAB) is the reagent incubated with hydrogen peroxide to generate the brown staining seen in many IHC experiments. This formation can be enhanced using different metal ions like copper, silver, nickel, gold, and cobalt. The metals can be added either during the DAB incubation or after. The timing needs to be determined on a case-by-case basis for best results.

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