Tetramethylbenizidine IHC Peroxidase

This 3,3'5,5’- tetramethylbenzidine (TMB) is a insoluble chromogen substrate for horseradish peroxidase detection systems for IHC, ICC and In Situ hybridization. This is ready to use one component system and gives aqua brilliant blue color with peroxidase. Neutral Red color can be used as a counterstain. This TMB solution contains TMB, 1.25 mMol L-1, Hydrogen Peroxide substrate, acetate buffer, pH 4.9, non-toxic proprietary stabilizers and precipitating agents.

Application: TMB-IHC chromogen is very sensitive. It is recommended that the peroxidase should be diluted (approx. 16X) compared to DAB used as chromogen, e.g. if peroxidase is diluted 1:100 for DAB, one should use approx. 1:1600 dilution of peroxidase when TMB is used. 1. After the peroxidase reaction, wash slides thoroughly with PBS or Tris-HCl saline containing 0.1 % Tween 20. DO NOT ADD THIMEROSAL, azide or phenolic compounds as preservative in any buffers. 2. Add TMB-IHC solution and incubate for 5-15 minutes at room temp. 3. Stop reaction by washing thoroughly with distilled or deionized water. 4. Counterstain with Neutral Red solution 30-60 seconds. 5. Rinse slides with distilled water, dehydrate with gradient of alcohols, xylene or any other clearing agent. 6. Mount slides with organic mounting medium or our Organo/Limonene mount (AR-6504) . 7. Brilliant blue color is observed with TMB.