Introduction

Citrate buffer antigen retrieval is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues[1-6]. When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. The result is weak or negligible levels of protein detection. Under elevated temperatures, citrate buffer has been shown to greatly improve detection of some proteins, although not all. Several antigen retrieval condition should be tested when optimizing stainng conditions.

Materials

 Citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0)

• Mix 1.92 g of anhydrous citric acid in 1000 ml of distilled water. Adjust the pH to 6.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
• Store at room temperature for up to 3 months; for extended storage, store at 4^oC.

Procedure

1. Deparaffinize and rehydrate tissue sections.
2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100^oC. Either citrate or sodium citrate solution can be used;  sodium citrate is often the buffer of choice.
3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution and to neutralize acidity.
6. Proceed with tissue blocking for immunohistochemistry.

References

1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov;41(11):1599-604. PubMed Abstract
2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61.PubMed Abstract
3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec;174(4):301-7.PubMed Abstract
5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid.Medicina (B Aires). 54(2):129-32.PubMed Abstract
6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604.PubMed Abstract