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-EDTA
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
Solutions and Reagents:
Citrate-EDTA Buffer (10mM Citric Acid, 2mM EDTA, 0.05% Tween 20, pH 6.2):
Citric acid (anhydrous) ----------------- 1.92 g
EDTA (Sigma, Cat# E-5134) ------------ 0.74 g
Distilled water --------------------------- 1000 ml
Mix to dissolve. Adjust pH to 6.2 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.
Deparaffinize sections in 2 changes of xylene, 5 minutes each.
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).
Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
Rinse sections in PBS Tween 20 for 2x2 min.
Block sections with for 30 minutes.
Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
Rinse sections with PBS Tween 20 for 2x2 min.
Block sections with peroxidase blocking solution for 10 minutes.
Proceed to standard immunohistochemistry protocol.
Note: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.
1. Sheriffs IN, Rampling D, Smith VV (2001) Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J Clin Pathol. 54(7):517-20. PubMed Abstract