Citrate Buffer Antigen Retrieval Protocol
Background for Citrate Buffer Antigen Retrieval
Formaldehyde fixation (2% or 4%, or as a component of 10% formalin) produces protein cross-links in tissues that tends to interfere with antibody penetration. This is particularly true of paraffin-embedded formaldehyde-fixed tissue sections, where the degree of inhibition is high. Since chicken IgY antibodies are larger than rabbit or mouse IgG’s, this becomes an even more important issue.
The citrate-based “antigen retrieval” protocol outlined below has been shown to improve chicken IgY antibody penetration into formaldehyde-fixed paraffin-embedded sections, and can increase the degree and intensity of immunoreactivity and immunostaining.
Reagents (NOTE: You can use either the Sodium Citrate or Citric Acid Buffers in step #3, below)
“Sodium Citrate Buffer” (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Weigh out 2.94 grams of trisodium citrate (dihydrate). Dissolve in approximately 900 mls of deionized, distilled water. Adjust the pH to 6.00 with 1.0 N HCl. Add 0.5 ml of Tween-20. Mix. Bring up the volume to 1.0 litres with water. Store this solution at room temperature for 3 months or at 4˚C for longer storage.
“Citric Acid Buffer” (10mM Citric Acid, 0.05% Tween 20, pH 6.0)
Weigh out 1.92 grams of citric acid (anhydrous). Dissolve in approximately 900 mls of deionized, distilled water. Adjust the pH to 6.0 with 1.0 N NaOH. Add 0.5 ml of Tween-20. Mix. Bring up the volume to 1.0 litres with water. Store this solution at room temperature for 3 months or at 4˚C for longer storage.
“Phosphate-Buffered Saline” [“PBS”, 10 mM Sodium phosphate-buffered (pH 7.2) isotonic (0.9%, w/v) saline solution]
“PBS Tween” (0.05% Tween 20 in PBS)
Ethanol (80%, 90%, 95%, 100%) diluted with water
Citrate Buffer Antigen Retrieval Procedure
(for use with paraffin-embedded sections):
- Deparaffinize tissue sections in 2 changes of xylene (5 minutes each).
- Hydrate in 2 changes of 100% ethanol (3 minutes each), 95% ethanol (1 minute), 90% ethanol (1 minute), 80% ethanol (1 minute). Rinse in distilled water.
- Pre-heat steamer or water bath with staining dish containing either Sodium Citrate Buffer or Citrate Buffer. Wait until temperature reaches 95-100 °C.
NOTE: Microwave or pressure cooker can be used as an alternative as a heating source.
- Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation times will vary).
- Remove the staining dish, and allow it to cool to room temperature (for 20 minutes or so).
- Rinse sections in PBS Tween twice for 2 minutes each time.
NOTE: The remainder of this protocol is meant to be a suggestion, and can be substituted with your regular immunostaining protocol.
- Block sections for 30 minutes with BlokHen® (Aves labs) diluted 1:10 with water.
- Perform avidin/biotin blocking, if necessary.
- Incubate sections with primary antibody at appropriate dilution in antibody dilution buffer overnight at 4 °C. Since chicken IgY antibodies are larger than mammalian IgG’s, this overnight incubation allows more time for antibody penetration into tissue sections.
- Rinse sections with PBS Tween 20 twice for 5 minutes each time.
- Incubate sections with HRP-labeled secondary antibody at appropriate dilution in
- Block sections with peroxidase blocking solution for 10 minutes.
- Rinse with PBS Tween 20 for three times for 5 minutes each time.
- Proceed to standard immunohistochemistry protocol.
- 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): 1599-1604.
- 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.
- Brown RW, Chirala R. (1995). Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8 (5): 515-20.
- 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 (4): 301-7.
- Pellicer EM, Sundblad A (1994). Antigen retrieval by microwave oven with buffer of citric acid. Medicina (B Aires). 54 (2): 129-32.
- 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.