Antigen retrieval and permeabilization for IHC

After sample preparation, two further steps may be required: antigen retrieval and permeabilization.

IHC application guide

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Antigen retrieval

The process of sample fixation can sometimes lead to protein cross-linking, which masks antigens and can restrict antigen-antibody binding. Antigen retrieval enables an antibody to access the target protein within the tissue. This step is not necessary if the fixation was mild. For example, if frozen tissues were fixed with alcohol, antigen retrieval isn’t required as alcohols do not mask epitopes.

Masked epitopes can be recovered using either enzymatic/proteolytic antigen retrieval (PIER) or heat-induced antigen retrieval methods (HIER). Whereas PIER uses proteases, such as proteinase K, trypsin, and pepsin, HIER uses heat (from either a microwave, pressure cooker, steamer, water bath, or autoclave) and a selection of buffers. For a comparison of HIER and PIER, see Table 2 below.

However, the optimal antigen retrieval technique is dependent on several factors, including the antigen, tissue, fixation method, and primary antibody. Some antigens require a combination of heating and enzyme digestion. To identify the best method for your specific antigen, we recommend testing two methods of HIER, for example, citrate buffer pH 6 and Tris-EDTA pH 9, and one or two methods of PIER, for example, proteinase K and trypsin.

Table 2. Comparison of HIER and PIER antigen retrieval methods

HIER

PIER

Advantages

Gentler epitope retrieval and more definable parameters

Useful for epitopes that are difficult to retrieve

Citrate buffers of pH 6 are often used but high pH buffers have been shown to be widely applicable for many antibodies. Optimal pH must be determined experimentally.

Typically, pH 7.4

Temperature

Approximately 95˚C

Typically, 37˚C

Incubation time

10-20 minutes (commonly, 20 minutes)

5-30 minutes (commonly, 10-15 minutes)

Buffer composition

Depends on pH required (pH is target-dependent, as shown in figure below). Popular buffers include sodium citrate, EDTA and Tris-EDTA

Neutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin

Precautions

Heating using a microwave can result in unbalanced epitope retrieval due to uneven heating. Boiling can also lift tissue off of the slide.

Excessive enzymatic retrieval sometimes damages tissue morphology.

Permeabilization for IHC

Permeabilization is required for the antibody to access the inside of cells to detect the target antigen. Such antigens include intracellular proteins and cytoplasmic epitopes of transmembrane proteins.

Solvents or detergents are typically used for permeabilization. Solvents are generally recommended for cytoskeletal, viral, and some enzyme antigens. Detergent permeabilization can significantly improve antibody access to antigens in the cytoplasm, on the cytoplasmic face of the plasma membrane, and soluble nuclear antigens. Depending on your antigen of interest, either harsh or mild detergents can be used. Harsh detergents, such as Triton™ X-100 or NP-40, can disrupt proteins, whereas mild detergents, such as Tween 20®, saponin, or digitonin, do not dissolve plasma membranes.

Table 3. Solvent and detergent guidelines

Solvents

Comments

Solvents

Acetone Methanol

Acetone fixation will also permeabilize Methanol fixation can be used to permeabilize but is not always effective

Detergents

Triton™ X-100 or NP-40

Use 0.1 to 0.2% in PBS for 10 min only

Tween 20®, saponin, digitonin and Leucoperm

Use 0.2 to 0.5% for 10 to 30 min