Tris-EDTA pH 9 antigen retrieval: recipe, protocol and when to use it

Formalin preserves tissue by cross-linking proteins through methylene bridges. Those cross-links also hide many epitopes from antibodies, so a formalin-fixed, paraffin-embedded section can stain weakly or not at all even with a good antibody. Heat-induced epitope retrieval (HIER) heats the dewaxed section in a buffer, partly reversing the cross-links and restoring antibody binding. The buffer’s pH and composition change how much each epitope recovers.

Store at room temperature for up to about three months, or at 4 °C for longer. Many labs make a 10X stock without Tween 20 and add it to the working solution.

For comparison, citrate buffer pH 6.0 is 10 mM sodium citrate (2.94 g of trisodium citrate dihydrate per litre) with 0.05% Tween 20, adjusted to pH 6.0.

1. Dewax sections in xylene and rehydrate through graded ethanol to water.
2. Pre-heat the Tris-EDTA buffer in your heating device.
3. Heat the slides in the buffer using one of the methods below, keeping the sections covered at all times.
4. Remove from the heat and let the slides cool in the buffer for 20 minutes at room temperature.
5. Rinse in water, then in wash buffer such as TBS or PBS with 0.05% Tween 20.
6. Continue with peroxidase blocking, protein blocking and the primary antibody.

No buffer suits every antibody. When validating a new antibody, stain serial sections of a positive control tissue with both buffers, and with no retrieval, and choose the condition with the strongest specific staining and the cleanest background. Datasheets for IHC-validated antibodies usually state the retrieval used in the validation images, which is the best starting point.

Retrieval exposes more than your epitope. Two things in the tissue itself can mimic a real signal, so block them after retrieval and before the primary antibody:

• Endogenous peroxidase. Red blood cells and granulocytes contain peroxidase that turns HRP substrates brown. Quench it with 3% hydrogen peroxide for about 10 minutes.
• Endogenous biotin. Kidney, liver and spleen are rich in biotin, and heat retrieval exposes more of it. Avidin or streptavidin detection then stains it everywhere.

An endogenous biotin blocking kit works in two steps: incubate with avidin to bind the tissue biotin, rinse, then incubate with free biotin to fill the avidin’s remaining binding sites so it cannot capture your biotinylated antibody. Ten to fifteen minutes each is typical. The alternative is a biotin-free polymer detection system, which avoids the problem entirely.

Finish with a protein block, such as normal serum from the secondary antibody’s host species, to cut non-specific binding.

Retrieval is one link in a chain: fixation, retrieval, blocking, antibody, detection and counterstain. The applications guide walks through the full IHC workflow and its controls, and the monoclonal vs polyclonal guide explains how clonality affects staining of fixed tissue.