To 5 ml of distilled or deionized water in a test tube, add two drop of reagent B buffer, mix well. Add one drop of reagent C chromogen, mix well. Add one drop of reagent S substrate, mix well. This ready to use reagent is good for several hours. The unused AEC solution can be discarded according to city, county, state, province or country’s regulations. After the peroxidase reaction, wash slide with buffer (this buffer should not contain any sodium azide because peroxidase is inactivated), and distilled/ deionized water. Add few drops of ready to use AEC reagent, incubate at RT or 37ºC for 5-10 minutes. Wash 2-5X with buffer, followed by rinsing with distilled/deionized water. Add counterstain compatible with AEC (we recommend hematoxylin counterstain for 30-60 seconds). Wash with tap water, buffer pH 7.4 or higher followed by rinsing with distilled/deionized water.
