Formalin-fixed, paraffin-embedded (FFPE) tissue sections must be treated to remove the paraffin (de-paraffinization, de-waxing) and unmask hidden or latent epitopes in preparation for immunohistochemical (IHC) staining. If these steps are not performed, the antibodies will not have complete access to antigens in the tissue and will be unable to bind to the correct epitopes.

De-paraffinizing (de-waxing) and rehydrating

The solvent xylene is typically used to remove all paraffin from the tissue sections once they have been attached to microscope slides. Prior to de-paraffinization, the slides are heated to 55°C for ten minutes to melt the wax.   The slides are then washed multiple times with xylene to solubilize and remove the paraffin. Next, the xylene is removed by graded washes with xylene and ethanol. Finally, the sample is rehydrated through graded concentrations 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 sample. From this point until the final mounting step prior to imaging, the slides should remain wet to avoid nonspecific antibody binding and high background staining in the sections

Because of the hazardous nature of xylene, less-toxic xylene alternatives are now commercially available to de-wax the sample. However, 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, the sample is then hydrated by graded washes of ethanol to water.


Antigen or epitope retrieval

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

HIER is the most common approach to antigen retrieval, and the temperature, pH and time of incubation are critical factors that must be optimized for proper antigen unmasking without causing morphological damage. Sodium citrate (10 mM, pH 6), EDTA (1 mM, pH 8) and Tris/EDTA (pH 9) buffers are commonly used for HIER in conjunction with heat (usually 95-100 °C) supplied by a hotplate, a microwave oven, a pressure cooker, or a vegetable steamer.

The proteolysis-based approach utilizes the protease activity of pronase, pepsin, ficin, trypsin, proteinase K, or other proteases to partially digest proteins to unmask the antigenic epitopes.  The key to antigen retrieval success with proteolytic enzymes is to optimize the final enzyme concentration, the incubation temperature, and the incubation time. Antigen retrieval in special circumstances can be performed using a combination of both approaches.  Also, there are many antigens that do not have to be retrieved prior to staining.  If we don’t explicitly say on our website that antigen retrieval is required, try the staining first without doing it.

While formaldehyde fixation usually requires antigen retrieval to unmask epitopes tied up in crosslinks, antigen retrieval may also improve the staining efficiency of sections fixed with non-aldehyde-based fixatives.


Recommended reading
  1. Hassel, J. and Hand, A.R. (1974). J. Histochem. Cytochem. 22 229-239.