Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections

Worldwide use of the antigen retrieval (AR) technique has served to enhance the value of immunohistochemistry (IHC) for routine surgical pathology and has provided promise of improved reproducibility of IHC staining. However, after the initial introduction of the AR method, many variations and 'improvements' have been proposed, including different heating methods, heating times, and buffer solutions, with strong advocates of different variations of the method. Our approach has been to seek to standardize the end result of AR and IHC staining, rather than the details of the technical procedure per se. This approach focuses on determining the AR method that leads to optimal results for each individual laboratory and each antibody; we refer to this optimal result as 'maximal retrieval.' To explore the possibility of standardization of IHC through achieving 'maximal retrieval,' we used a 'test battery' that examines the principal technical variables for archival paraffin tissue sections. Tests were conducted using monoclonal antibodies to AE1, MIB1, p53 (Pab-1801, DO7, and BP53-12-1) for AR-IHC on formalin-fixed, paraffin-embedded tissues of renal carcinoma and breast carcinoma, that had been fixed in formalin for different periods: 4, 12, and 24 hours and 3, 7, 14, and 30 days. Using the 'test battery' approach, varying the buffer solution, pH, and heating time, an optimal protocol of AR could be established. For MIB 1, Tris-HCl buffer of pH 1 yielded maximal retrieval for various tissues tested; for all other antibodies tested, both Tris-HCl buffer of pH 1 and pH 10 may yield maximal retrieval results for all tested tissue sections. Establishing optimal or maximal retrieval is a significant step toward the standardization of IHC on archival formalin paraffin tissues.