Abstract
The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, D'amanto L, Sabarrini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed paraffinembedded tissue sections. J Pathol 171: 83–98.
Clayden J, Greeves N, Warren S, Wothers P (2001) Organic Chemistry.Oxford, United Kingdom: Oxford University Press, pp. P293–P294.
Evers P, Uylings HBM (1994) Microwave-stimulated antigen retrieval is pH and temperature dependent. J Histochem Cytochem 42: 1555–1563.
Kim SH, Jung KC, Shin YK, Lee KM, Park YS, Choi YL, Oh KI, Kim MK, Chung DH, Song HG, Park SH (2002) The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine. Histochem J 34: 97–103.
Kim SH, Shin YK, Lee KM, Lee JS, Yun JH, Lee SH (2003) An improved protocol of biotinylated tyramine-based immunohistochemistry minimizing nonspecific background staining. J Histochem Cytochem 51: 129–132.
Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate mediated high temperature antigen retrieval. J Pathol 174: 301–307.
Morgan JM, Navabi H, Jasani B (1997) Role of calcium chelation in high-temperature antigen retrieval at different pH values. J Pathol 182: 233–237.
Norton AJ, Jordan S, Yeomans P (1994) Brief, high-temperature heat denaturation (pressure cooking): A simple and effective method of antigen retrieval for routinely processed tissues. JPathol 173: 371–379.
Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, Damiani S, Ercolessi C, Sanri F, Pieri F, Leoncini L, Falini B (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183: 116–123.
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39: 741–748.
Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43: 193–201.
Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: Past, present, and future. J Histochem Cytochem 45: 327–343.
Stoll VS, Blanchard JS (1990) Buffers: Principles and practice. In: Deutscher MP, eds. Guide to Protein Purification. Vol. 182. Methods in Enzymology, 1st edn. San Diego: Academic press, pp. 24–38.
Taylor CR, Shi SR, Cote RJ (1996a) Antigen retrieval for immunohistochemistry: Status and need for greater standardization. Appl Immunohistochem 4: 144–166.
Taylor CR, Shi SR, Chen C, Young L, Cote RJ (1996b) Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer. Biotechnic Histochem 71: 263–270.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kim, S.H., Kook, M.C., Shin, Y.K. et al. Evaluation of Antigen Retrieval Buffer Systems. Histochem J 35, 409–416 (2004). https://doi.org/10.1023/B:HIJO.0000039854.17808.e0
Issue Date:
DOI: https://doi.org/10.1023/B:HIJO.0000039854.17808.e0