Dominik Haudenschild, from C.C. Haudenschild, November 8, 1994

It is unknown exactly how or why antigen retrieval works, but a theory suggests that certain cross-links or methylene bridges are broken down thus exposing more antigenic sites to the primary antibody. The high temperature is not deleterious and is in fact required to complete the AR process.

Antigen Retrieval techniques will enhance the immunohistochemical response of many antigens that have been fixed in formalin or other cross-linking or precipitating fixatives. However, frozen sections fixed in acetone or alcohol do not require antigen retrieval. Frozen sections fixed in formalin can be enhanced by AR techniques to a degree.

Microwave oven of at least 750 watts on High.

APES/TESPA or Poly-L-Lysine treated slides.

Stock solution A = 0.1M solution of Citric acid

Stock solution B = 0.1M solution of Sodium Citrate

Working AR solution: Add together 9ml solution A with 41ml solution B and bring to 500ml with Water. Adjust pH to 6.0

Phosphate buffered Saline solution, pH 7.4 to 7.6

Tupperware style bowl, large enough to hold 1 quart

Deparaffinize and carry sections through 3 changes of 100% alcohol.

Block endogenous Peroxidase with 0.3% Hydrogen peroxide in absolute methanol for 30 minutes

Place slides in a plastic rack and immerse in a heat-resistant plastic dish containing 500ml of working AR solution.

Heat ion the microwave oven for 15 minutes. Boiling will occur, and is allowable.

Remove container from the microwave, and allow to cool down fro about twenty minutes to room temperature.

Rinse in 2 changes of Water, followed by two changes of PBS

At this time, you may continue the immunohistochemistry procedure at the serum blocking step.

The boiling step may cause fatty tissue sections to become dislodged from the slides. It has been found that substituting a waterbath capable of maintaining the AR solution at 95°C fro 20 minutes eliminates the harsh scrubbing action of boiling solution and gives comparable results.

Antigen Retrieval is used in lieu of trypsinization and in fact sections stain more intensely by virtue of exposure of more antigenic sites after AR than after trypsinization.

Primary antibody concentrations will usually have to be optimized for use after AR. Example: A 1:100 may requires more dilute concentration of 1:500 with AR.

This method is superior to the "Heavy Metal" type of antigen retrieval method and is much safer as the solution may be poured down the sink and there is no worry of toxic fumes or spills.

Shi et al. Antigen Retrieval in Formalin-Fixed, Paraffin Embedded Tissues. Journal of Histochemistry/Cytochemistry 39 (6) 741-748, 1991

AMAC, Inc. Information leaflet on Retrieval of ER and KI-67 utilizing citrate buffer. 1992

Communications with C.E. Lincoln, Holland Lab, American Red Cross Experimental Pathology Laboratory of Christian Haudenschild.