Formalin-fixed, paraffin-embedded tissue sections must be treated to remove the paraffin and unmask the antigen epitopes in preparation for immunohistochemistry (IHC) staining. If these steps are not performed, the antibodies will not have complete access to the tissue and will be unable to bind to the correct epitopes.

Learn more

  • Overview of Immunohistochemistry (includes links to individual pages on all aspects and stages of IHC)

  • Deparaffinizing (Clearing) and Rehydrating

    Xylene is used to remove all paraffin from the tissue sample to give the antibodies complete access to the tissue. Before clearing, the samples are heated to 55°C for ten minutes to melt the paraffin and then washed multiple times with xylene to remove the paraffin. Next, the xylene is removed by graded washes with xylene and ethanol, and then the sample is rehydrated through graded washes of ethanol in water, ending in a final rinse in pure water (see graph below). The number of washes and the reagent concentrations must be optimized for each antigen and tissue saple. From this point until final mounting, the slides should remain wet to avoid nonspecific antibody binding and high background staining.

    Because of the hazardous nature of xylene, commercial, less-toxic xylene alternatives are now commercially available to clear the sample. The efficacy of these alternatives in removing all paraffin from the sample, which is critical for optimum target antigen detection, can be variable.

    Graphic representation of washes used to clear and rehydrate IHC samples. Paraffin is removed from IHC tissue sample by consecutive washes with xylene. Xylene is then removed with graded washes of xylene to ethanol, and the sample is then hydrated by graded washes of ethanol to water.

    Antigen Retrieval

    Formaldehyde forms methylene bridges between proteins, which can hinder epitope recognition by the primary antibodies. Two methods to remove these bridges are heat-induced epitope retrieval (HIER) and proteolytic-induced epitope retrieval (PIER).

    HIER is the most common approach to antigen retrieval, and temperature, pH and time of incubation are critical factors that must be optimized for proper antigen unmasking without causing morphological damage. Sodium citrate (pH 6) and Tris/EDTA (pH 9) buffers are commonly used with HIER in conjunction with the heat source (microwave oven, pressure cooker or vegetable steamer).

    The PIER approach utilizes the enzymatic activity of pronase, pepsin, ficin, trypsin or proteinase K to partially digest proteins to unmask the antibody epitopes, and the efficacy of using PIER is dependent upon enzyme concentration and incubation time. Antigen retrieval can be performed using either HIER or PIER, or through a combination of both approaches.

    While formaldehyde fixation usually requires this procedure to unmask epitopes due to methylene crossbridges, antigen retrieval may also improve the antigenicity of alcohol-fixed sections.