Antigen Retrieval Buffer, Citrate or Tris-EDTA

Formalin and paraformaldehyde fixation cross-link proteins and mask epitopes, which weakens IHC and immunofluorescence staining or causes false negatives. Heating sections in a retrieval buffer reverses those cross-links and restores the epitopes. The buffer's chemistry and pH decide how strong that retrieval is.

Citrate pH 6.0 (10 mM citric acid and sodium citrate at working strength) is the gentlest. It is recommended for tissues with high background, such as brain, spinal cord, and liver, and for membrane and cytoplasmic proteins.

Tris-EDTA pH 8.0 and pH 9.0 (1 mM EDTA at working strength) retrieve more strongly than citrate. They are recommended for tissues with low background, such as heart, spleen, and intestine, and for nuclear antigens. For both monoclonal and polyclonal primary antibodies, retrieval strength runs Tris-EDTA pH 9.0, then Tris-EDTA pH 8.0, then citrate.

Each buffer comes two ways. The 20× concentrate in a 250 mL bottle mixes 10 mL with 190 mL of distilled water for 200 mL of working buffer, so one bottle makes 5 L. The 1 L ready-to-use bottle pours straight into the retrieval chamber.

Use them with the ConductScience heat-induced antigen retrieval system, which heats pH 6 to 9 buffers directly for up to 96 slides, or with the heating method your protocol specifies. The buffers store and ship at room temperature and keep for 12 months. Use diluted buffer the same day or store it briefly at 4 °C, and discard any buffer that turns yellow or cloudy.