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1. Dr. Usman Shams
Chromogens Used in IHC

2. 3,3'-Diaminobenzidine (DAB)
 As its water-soluble tetrahydrochloride, it has been used in
immunohistochemical staining of nucleic acids and proteins.

3.  In research, this reaction is used to stain cells that were
prepared with hydrogen peroxidase enzyme, following
common immunocytochemistry protocols. In research being
done onAlzheimer's Disease,Aβ protein amyloid plaques are
targeted by a primary antibody, and subsequently by a
secondary antibody, which is conjugated with a peroxidase
enzyme.This will bind DAB as a substrate and oxidize it,
producing an easily observable brown color.

4. Characteristics of common chromogenic reporters used for IHC
Enzyme Label Substrate Reporter color
Horseradish peroxidase
(HRP)
3,3'-diaminobenzidine (DAB) Brown to black
Alkaline phosphatase (AP) Combination of nitro blue
tetrazolium chloride (NBT)
and 5-bromo-4-chloro-3-
indolyl phosphate (BCIP)
Black to purple
Glucose oxidase Nitro blue tetrazolium
chloride (NBT)
Blue to purple
β-galactosidase 5-bromo-4-chloro-3-indoyl-
β-D-galactopyranoside (BCIG
or X-Gal)
Blue

5. Substrates for immunohistochemistry.
Substrate Format Enzyme Features Sensitivity Signal color
DAB Dry powder HRP Can be
formulated
Medium Brown
Metal-enhanced
DAB
2-component
reagent kit
HRP 50X more
sensitive than
DAB alone
Highest Brown to black
BCIP Dry powder AP Can be
formulated
Medium Blue to purple
NBT Dry powder AP and glucose
oxidase
Non-
carcinogenic
Medium Blue to purple
1-step
NBT/BCIP
Single step,
ready-to-use
AP Low
background for
high sensitivity
High Black to purple
1-step
NBT/BCIP +
suppressor
Single step,
ready-to-use
AP Contains
levamisole for
endogenous
peroxidase
inhibition
High Black to purple