Proteolytic Antigen Retrieval

Antigenic determinants masked by formalin-fixation and paraffin-embedding may also be exposed by enzymatic digestion. This can, however, not be used with frozen sections or cells which are not paraffin-embedded. The beneficial effects of protease treatment are presumably related to cleavage of the molecular cross-links by the 

---

It is possible to restore antigenicity in tissue sections by digesting the rehydrated sections with any of several proteolytic enzymes, or by incubating the tissue section at elevated temperatures in any of several liquids, including water and various buffers (antigen retrieval).12... 

Hazelbag, H. M., V. D. Broek, L. J. C. M., Van Dorst, E. B. L., Offerhaus, G. J. A., Fleuren, G. J., and Hogendoom, P. C. W. 1995. Immunostaining of chain-specific keratins in formalin-fixed, paraffin-embedded tissues a comparison of various antigen retrieval systems using microwave heating and proteolytic pretreatments. J. Histochem. Cytochem. 43 429-437. 

The process of antibody optimization also requires that different temperatures of antigen retrieval be explored, and although citrate buffer at 10 mMol/litre is a good universal retrieval solution, a higher pH may be required for more capricious antigens as previously discussed (Shi et al, 1995). In addition, the synergistic action of proteolytic digestion should be routinely explored as a pre-MW and post-MW procedure. 

Demasking protocol is inappropriate or has been omitted. Some tissue antigens require proteolytic enzyme digestion or heat induced antigen retrieval performed prior to staining. The need for pretreatment depends on the type and extent of fixation, specific characteristics of the antigen and the type of antibody used. Use the pretreatment method recommended by the manufacturer. No single pretreatment is suitable for all applications. 29-33, 51-65... 

DAKO TRS (Dako Corp., Carpinteria, CA) is an excellent alternative retrieval buffer. For some antigens, 0.05 M Tris buffer (pH 10.0) is useful. This buffer is highly proteolytic therefore, if tissue deterioration occurs, reduce the concentration of buffer and/or the heating time, and/or employ a hot start method as described in Subheading 3.3. 

---