-Histofine®
DAB-3S kit
Peroxidase Chromogen/Substrate kit
Reagent supplied ：NICHIREI BIOSCIENCES INC.
6-19-20, Tsukiji, Chuo-ku, Tokyo, 104-8402, JAPAN
Phone: 81-3-3248-2207 Facsimile: 81-3-3248-2243

500 tests

Code : 415192F

1500 tests

Code : 415194F

3,3’-DIAMINOBENZIDINE TETRAHYDROCHLORIDE (DAB)
SUBSTRATE KITSUBSTRATE FOR HORSERADISH PEROXIDASE
FORM

Liquid.
This product contains the following reagents;
Reagent A : DAB solution concentrate
Reagent B : Enhancer solution concentrate
Reagent C : Hydrogen peroxide solution concentrate
Dropping bottle

…
…
…
…

[500 tests]
3mL×1 bottle
3mL×1 bottle
3mL×1 bottle
1 bottle[1500 tests]
9mL×1 bottle
9mL×1 bottle
9mL×1 bottle
1 bottle

PREPARATION of Chromogen/Substrate solution

1.Add 1 drop of DAB solution concentrate(Reagent A) to 1 mL of distilled water into the tube. Mix well.
2.Add 1 drop of Enhancer solution concentrate (Reagent B) to 1 mL of diluted Reagent A solution. Mix again.
3.Add 1drop of Hydrogen peroxide solution concentrate (Reagent C) to 1 mL of diluted Reagent A and Reagent Bsolution. Mix again.
4. The final mixture is stable for 14 days at 2-8°C with light shielding*.
*: Though the final mixture is stable for 14 days at 2-8 °C with light shielding, the mixture may result in a precipitate with time.
The precipitation will have no effect on staining. However, in some cases, some precipitation attached on slides or tissues, may
disturb getting crisp image. To avoid such precipitation attachment, following procedures are recommended.
1. Use supernatant of the mixture
2. Rinse the slides in PBS containing 0.1% Tween 20 or TBS containing Tween 20 at room temperature for three minutes, intwice, before and after the step of the mixture reaction described in USAGE.
Note regarding Dropping bottle
1) The dropping bottle may be suitable for dropping the final mixture on the slides, not for mixing the reagents, nor for storage of the final mixture.
2) If the final mixture is moved from the tube to the dropping bottle, careful handling (e.g. using pipette) is required.
3) If the remaining final mixture is stored, it shall be moved from the dropping bottle to appropriate vessels (e.g. sealed bottles or tubes) and stored at
2 – 8 °C with light shielding.
4) Rinse the dropping bottle thoroughly with distilled water after use and dry it enough prior to reuse.

USAGE

This substrate will produce a brown colored deposit upon reaction with peroxidase.
For color development, incubate the tissue section, which has been reacted with peroxidase conjugate,with 1-2 drops of the final mixture at room temperature for 5-20 minutes. Color development may bemonitored with a microscope. After adequate coloration, wash the slide with tap water.
Note regarding the final mixture : Use the final mixture after adjusting to room temperature.

This brown deposit is insoluble in organic solvent. Therefore, the slide can be mounted withpermanent mounting media after washing with water, dehydrated in graded series of alcohol andcleared in xylene.

STORAGEStore at 2-8°C.

REMARKSFor research use only. Not for diagnostic use.

WARNING !

1.Reagent A (DAB solution concentrate) should be handled with care for it contains carcinogen.
2.Reagent C (Hydrogen peroxide solution concentrate) should be handled with care for it contains
Hydrogen peroxide.

-Histofine® is NICHIREI BIOSCIENCES INC’s registered trademark. The registration countries would be referred to us.

01/2008