Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

Solutions and Reagents:  

Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):

• Tri-sodium citrate (dihydrate) --------- 2.94 g
• Distilled water ------------------------- 1000 ml

Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

Note: this buffer is commonly used and works perfectly with many antibodies. It gives very nice intense staining with very low background.

Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):

• Citric acid (anhydrous)  --------------- 1.92 g
• Distilled water -------------------------- 1000 ml

Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

Procedure:

1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.
3. Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 °C.
4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).
5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
6. Rinse sections in PBS Tween 20 for 2x2 min.
7. Block sections with for 30 minutes.
8. Perform avidin/biotin blocking if necessary.
9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
10. Rinse sections with PBS Tween 20 for 2x2 min.
11. Block sections with peroxidase blocking solution for 10 minutes.
12. Rinse with PBS Tween 20 for 3x2 min.
13. Proceed to standard immunohistochemistry protocol.

Note: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.

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