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3,3-Diaminobenzidine (DAB) Liquid Substrate
System
Catalog Number D7304
Storage Temperature 2–8 C
Product Description
The DAB (3,3-Diaminobenzidine tetrahydrochloride)
Liquid Substrate System has been developed for use in
immunohistological and immunoblotting procedures as
a precipitating substrate for the detection of peroxidase
activity. DAB is the immunohistological substrate of
choice because it produces an intense brown stain that
is easily observed visually. The end product is resistant
to alcohol, therefore, a variety of counterstains and
mounting media can be used with the DAB Liquid
Substrate System. The DAB Liquid Substrate System
provides all the chromogen and buffer/peroxide
solutions needed to produce a fast and convenient DAB
substrate solution. The DAB Liquid Substrate System
is not recommended for ELISA (multiwell) procedures.
Components
The DAB Liquid Substrate System consists of the
following reagents:
DAB Liquid Buffer 225 mL
(Catalog Number D7429)
10 DAB Liquid Chromagen 25 mL
(Catalog Number D7554)
The DAB Liquid Substrate System provides reagents
sufficient to prepare 250 mL of DAB Substrate Solution.
Equipment and Reagents Required but Not
Provided
Test Tubes
Pipette capable of delivering 1 mL
Graduated cylinder to measure 9 mL
Whatman
®
Puradisc 30 syringe filter units,
disposable, cellulose acetate, pore size 0.2 m,
(Catalog Number WHA10462701)
Nickel(II) chloride hexahydrate (NiCl
2
6H
2
O,
Catalog Number 223387) or Cobalt(II) chloride
hexahydrate (CoCl
2
6H
2
O, Catalog Number
202185), 0.3% (w/v) solution for enhancement of
tissue stains
Tris buffered saline (TBS, Catalog Number T5030)
for washing
Precautions and Disclaimer
This product is for Research Use Only. Not for Use in
Diagnostic Procedures. Please consult the Safety Data
Sheet for information regarding hazards and safe
handling practices.
Preparation Instructions
Prepare 10 mL of DAB Substrate Solution by adding
1 mL of 10 DAB Liquid Chromogen (Catalog Number
D7554) to 9 mL of DAB Liquid Buffer (Catalog
Number D7429). Mix well. For best results, use the
solution immediately.
DAB reactions may be enhanced by the addition of
NiCl
2
or CoCl
2
to the DAB Substrate Solution. Add
1 mL of a 0.3% (w/v) metal salt solution to 10 mL of
DAB Substrate Solution. The addition of a metal salt to
DAB changes the color from brown to black or blue-
black.
Occasionally DAB solutions may be hazy. The
haziness may be removed by filtering the DAB solution
through a 0.2 m filter.
Storage/Stability
The product ships on wet ice and storage at 2–8 C is
recommended. The reagents are stable for 12 months
after manufacture. See product labels for actual
expiration date.
Procedure
1. Cover tissue sections with 0.2–0.5 mL of the DAB
Substrate Solution.
2. DAB is a fast-reacting substrate. Monitor color
development carefully during the reaction to
prevent overdevelopment and high background.
The reaction may be stopped by gently washing the
slide in water or TBS.
3. When finished, dispose of any remaining DAB
Substrate Solution in a manner consistent with
proper hazardous material handling protocols for
your institution.
2
Troubleshooting
Background too high
1. Prior to the application of the primary antibody,
block the tissue with 10% (v/v) normal serum from
the host species of the second antibody.
2. Prior to antibody incubations, block endogenous
peroxidase by flooding the slides with a solution of
4 parts methanol to 1 part 3% H
2
O
2
solution.
3. Decrease the staining time.
4. Titer the conjugate to optimize the working dilution.
No color develops or color is too faint
1. Adjust the concentration of the primary antibody.
2. Adjust the concentration of the secondary antibody.
3. Determine if the enzyme conjugate is active.
4. Consider using an amplifying system such as
avidin-biotin.
5. Increase the staining time.
6. Determine if enzymatic treatment (unmasking) of
the antigen is required prior to application of the
primary antibody.
References
1. Nakane, P.K., and Pierce, G.B., Jr., J. Histochem.
Cytochem., 14(12), 929-931 (1966).
2. Trojanowski, J.Q. et al., J. Histochem. Cytochem.,
31, 1217-1223 (1983).
3. DeJong, A.S.H. et al., Histochem. J., 17(10), 1119-
1130 (1985).
4. Chu, N.M. et al., J. Histochem. Cytochem., 37(2),
257-263 (1989).
5. Merchenthaler, I. et al., “Silver Intensification in
Immunocytochemistry”, in Techniques in
Immunocytochemistry (Bullock, G., and Petrusz, P.,
eds.). Academic Press Ltd. (San Diego, CA: 1989),
pp. 217-252.
6. Hsu, S., and Soban, E., J. Histochem. Cytochem.,
30(10), 1079-1082 (1982).
Whatman is a registered trademark of GE Healthcare.
VNC,GCY,MAM 11/18-1
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