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Protocol for AEC Peroxidase Substrate Solution
AEC (3-amino-9-ethylcarbazole) Peroxidase Substrate Solution – Red
Final Dilution:
0.05% AEC and 0.015% H2O2 in 0.05 M Acetate Buffer, pH 5.5
Stock Solutions:
1% AEC (20x) in 100% DMF (N, N Dimethylformamide):
Dissolve 0.1 g of AEC in 10 ml
of 100% DMF and store this solution at
4
°C.
0.3% H2O2 (20x) in Distilled Water:
Add 100 ul of 30% H2O2 in 10 ml distilled water and store at 4 °C.
1M Acetate Buffer (20x), pH
5.5:
Sodium acetate, trihydrate (MW
136.1) ------ 13.6 g
Distilled water
----------------------------------- 100 ml
Mix to dissolve and adjust pH to 5.5 using concentrated HCl or glacial acetic acid.
0.05M Acetate Buffer (1x), pH 5.5:
1M Acetate buffer ------------------------------- 1 ml
Distilled water ----------------------------------- 19 ml
Mix well and adjust pH if necessoary.
Working Solution:
Add 5 drops of 1% AEC to 5 ml
of 0.05M Acetate Buffer, pH5.5 and mix well.
Add 5 drops of 0.3% H2O2 and mix well.
Incubate sections for 10-15 minutes at room temperature.
Note: AEC color products can be washed off with ethanol/alcohol solution. So slides must be coverslipped using aqueous mounting media.