1. Place five-micron thick tissue sections on glass slides coated with poly L-lysine or APTES.
2. Deparaffinize and rehydrate sections as usual.
3. Place slides in a Coplin jar containing 10mM sodium citrate buffer, pH 6.0; cover with a vented plastic wrap and place the jar in a steamer (Black & Decker™) for 20 minutes.
4. Take out the jar and let the sections cool in the jar for 20 minutes at room temperature. THIS STEP IS VERY CRITICAL AND SHOULD NOT BE AVOIDED.
5. Wash sections in buffer for 2 x 5 minutes.
6. Block endogenous peroxidase as usual.
7. Wash sections in buffer for 2 x 5 minutes.
8. Block nonspecific sites with normal serum as usual.
9. Place optimally diluted primary antibody on the sections (incubation time and temperature for a given set of experimental conditions should be determined by the investigator).
10. Wash sections in buffer for 2 x 5 minutes.
11. Remaining procedure is same as routinely performed in your laboratory.