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Universal
TM
One-step Immunohistochemistry (IHC) Kit
(For R&D Use only)
Product Name & Catalog Number:
Product Name
UniversalTM One-step IHC Kit-R
With DAB Substrate
(Suitable for rabbit primary
antibodies)
UniversalTM One-step IHC Kit-M
With DAB Substrate
(Suitable for mouse primary
antibodies)
UniversalTM One-step IHC Kit-G
With DAB Substrate
(Suitable for goat primary
antibodies)
UniversalTM One-step IHC Kit-R
With BCIP/NBT Substrate
(Suitable for rabbit primary
antibodies)
UniversalTM One-step IHC Kit-M
With BCIP/NBT Substrate
(Suitable for mouse primary
antibodies)
UniversalTM One-step IHC Kit-G
With BCIP/NBT Substrate
(Suitable for goat primary
antibodies)
Catalog #
Size
Content
IHC-R60
1 kit
(For 100 slides)
IHC-M60
1 kit
(For 100 slides)
IHC-G60
1 kit
(For 100 slides)
IHC-R61
1 kit
(For 100 slides)
IHC-M61
1 kit
(For 100 slides)
IHC-G61
1 kit
(For 100 slides)
IHC Pretreatment Solution (20ML),
IHC Enhancer (20ML),
10X IHC Probe-R0 (2ML),
10X Rapid Wash Solution (10ML),
100X DAB Solution Set (1ML each),
User Manual (One copy).
IHC Pretreatment Solution (20ML),
IHC Enhancer (20ML),
10X IHC Probe-M0 (2ML),
10X Rapid Wash Solution (10ML),
100X DAB Solution Set (1ML each),
User Manual (One copy).
IHC Pretreatment Solution (20 ML),
IHC Enhancer (20 ML),
10X IHC Probe-G0 (2ML),
10X Rapid Wash Solution (10 ML),
100X DAB Solution Set (1ML each),
User Manual (One copy).
IHC Pretreatment Solution (20ML),
IHC Enhancer (20ML),
10X IHC Probe-R1 (2ML),
10X Rapid Wash Solution (10ML),
BCIP/NBT Substrate (20ML),
User Manual (One copy).
IHC Pretreatment Solution (20ML),
IHC Enhancer (20ML),
10X IHC Probe-M1 (2ML),
10X Rapid Wash Solution (10ML),
BCIP/NBT Substrate (20ML),
User Manual (One copy).
IHC Pretreatment Solution (20 ML),
IHC Enhancer (20 ML),
10X IHC Probe-G1 (2ML),
10X Rapid Wash Solution (10 ML),
BCIP/NBT Substrate (20ML),
User Manual (One copy).
.
Product Description:
Allied Biotech, Inc
10075 Tyler Place, Suite 19, Ijamsville, MD 21754
Tel: (301) 874-0495; Fax: (240) 465-5802
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TM
Universal One-step IHC is a latest breakthrough technology (patent pending) for performing Immunohistochemistry
(IHC) or Immunocytochemistry (ICC). Unlike regular (classical) IHC/ICC, the One-step IHC/ICC technology combines
blocking, primary antibody staining, washing and secondary antibody staining into a rapid one-step reaction. Therefore,
using UniversalTM One-step IHC kit, your IHC/ICC becomes so simple and is no longer a time-consuming and labor-intensive
procedure. The One-step IHC kit is a universal kit, which is suitable for most primary antibodies and no need of secondary
antibody. Currently, we provide six types of UniversalTM One-step IHC kits as shown on the above table. Each kit is sufficient
to stain 100 slides or 400 wells for 96-well plates.
Application:
Immunohistochemistry (IHC) or Immunocytochemistry (ICC).
Features:
Easy, a simple One-step reaction instead of the 4-step procedure: blocking, primary antibody-binding, washing and
2nd antibody-binding.
Rapid, just 1~2 hrs instead of 4~5 hrs.
Universal, suitable for most primary antibodies and no need of secondary antibody.
Sensitive, comparable sensitivity with regular IHC or ICC.
Reproducible, highly reproducible results.
Storage:
 Pretreatment Solution and Enhancer: for continuous use, store in 2~8 °C for up to 1 month, or store in -20 °C for up to a year.
 IHC Probe and Substrate: for continuous use, store in -20 °C for up to 6 months or store in -70 °C manual defrost freezer for
up to a year. Repeated freezing and thawing is not recommended.
 Rapid Wash Solution: store in room temperature for up to one year.
Note: Kit will be shipped in ambient temperature.
Comparison of One-step IHC with Regular IHC:
Protocol:
I.

Other required materials:
Primary antibody: Purified monoclonal or affinity-purified polyclonal antibodies (antigen-specific IgGs) are
preferred. Although unpurified antibody or serum can also be used for the One-step IHC, user may need to
optimize staining conditions to avoid non-specific binding.
Note: We currently provide six types of UniversalTM One-step IHC kits. Kits labeled with -R are suitable for using together
with rabbit primary antibodies, with -M suitable for using together with mouse primary antibodies, and with -G suitable for using
together with goat primary antibodies.

Fixatives: 30% Buffer-neutralized Formalin or Z-fix.

Counter staining Reagent: Mayer’s hematoxylin or other proper counter staining reagents.
Allied Biotech, Inc
10075 Tyler Place, Suite 19, Ijamsville, MD 21754
Tel: (301) 874-0495; Fax: (240) 465-5802
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II.
Reagent preparation:

One-step IHC Antibody Mixture: Mix 5-30ul of primary antibody and 100ul IHC Probe (supplied in the kit) with
1ml of IHC Enhancer (supplied in the kit). The amount of primary antibody added may vary depending on the quality
of the antibody and it may need optimization by user to obtain best results.

1X Rapid Wash Solution: Dilute 10ml of 10X Rapid Wash Solution (supplied in the kit) with 90ml distilled water.

1X DAB Mixture: Mix 100ul of 100X DAB solution (supplied in the kit), 100ul of 100X DAB buffer (supplied in the
kit) with 10 ml of distilled water.
III. Procedure:
1. Fix: Incubate sample (tissue or cells) on slide with Formalin (or other proper fixatives) for 20 min and then rinse
the slide with 600ul PBS. Note: this step is same to the method for regular IHC or ICC.
2. Pretreatment: Incubate the pre-fixed tissue section or cells on slide with 200ul (per slide) of Pretreatment
Solution for 10 minutes at room temperature in order to inactivate the endogenous peroxidase, and then rinse this
slide once with 600ul PBS.
3. One-step Reaction: Incubate the above slide (from Step 2) with 200ul of the prepared One-step IHC Antibody
Mixture (a mixture of primary antibody, IHC enhancer and IHC Probe) for 30~60 minute at room temperature.
4.
Wash: Rinse the slide three times with 600ul of 1X Rapid Wash Solution and once with 2ml of PBS.
Development: Apply 200ul of the pre-prepared 1XDAB mixture (for kits IHC-R60, IHC-M60 and IHC-G60) or
IHC-R61, IHC-M61 and IHC-G61) to the washed section on slide and incubate
for approximate 2 min or an appropriate time until a desired color appears. To stop the development, simply rinse
slide with distilled water.
5.
BCIP/NBT substrate mixture (for kits
6. Counter-staining: Incubate the slide with the solution of Mayer’s hermatoxyin for 0.5-5 min at room temperature
until a desired background color appears. Rinse the slide with distilled water. Note: this step is same to the method
for regular IHC or ICC.
7. After Staining: Take photos and/or mount the section with glycerol gelatin and a sealing cover-slip with clear nail
polish. Note: This step must be performed according to the standard procedure used for regular IHC or ICC
Example:
Comparison of One-step ICC with Regular ICC in Detection of the Expression of a Flag-tagged protein in A549 Cells. A549
cells cultured in 96-well plate were directly fixed on the wells of tissue culture plate and then pretreated with the kit-provided pretreatment
solution. Expressed Flag-tagged proteins in cells were detected using mouse anti-Flag antibody by a regular ICC procedure (A) or by a
one-step IHC procedure (B) or by a one-step IHC procedure but without using primary antibody (C). As showed in the picture, the results
by both methods are very similar but the testing time and procedure are greatly different. A: 5 hours (using regular method). B: <1 hour
(using One-step IHC kit). C: One-step ICC without primary antibody (as a negative control). Note: the brown color signals indicated the
positive stains using anti-Flag antibody and DAB substrate.
Allied Biotech, Inc
10075 Tyler Place, Suite 19, Ijamsville, MD 21754
Tel: (301) 874-0495; Fax: (240) 465-5802
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Troubleshooting:
Problem
Probable Cause
Weak Signal
or No
Staining
Inadequate deparaffinization
Solution
Deparaffinize sections longer or change fresh xylene
Inactive primary antibodies
Replace with a new batch of antibodies
Antibodies do not work due to improper
storage
Aliquot antibodies into smaller volumes and store in freezer (-20 to 70 C) and avoid repeated freeze and thaw cycles. Or store
antibodies according to manufacture's instructions.
Increase the concentration of primary and/or One-step IHC probe. Or
Concentration of primary antibody or IHC run a serial dilution test to determine the optimal dilution that gives
probe was too low
the best signal to noise ratio
Incorrect primary antibody type
Check the label of IHC Kit and select compatible primary antibodies
and One-Step IHC Kit. Kits labeled with -R are suitable for rabbit
primary antibodies, with -M suitable for mouse primary antibodies,
and with -G suitable for goat primary antibodies.
Poor specificity of primary antibodies.
Use purified monoclonal or affinity-purified polyclonal primary
antibodies.
Inadequate antibody incubation time
Increase antibody incubation time
Inadequate or improper tissue fixation
Increase duration of postfixation or try different fixatives
Tissue overfixation
Reduce the duration of postfixation. If the tissue has already been
overfixed, perform an appropriate or recommended antigen retrieval
antigen retrieval procedure.
Defective One-step IHC probe
Replace with a new batch of One-step IHC probe.
Inadequate substrate incubation time
Increase the substrate incubation time.
Over-staining The concentration of primary and/or One- Reduce the concentration of antibody or IHC probe, or perform a
step IHC probe was too high
titration to determine the optimal dilution for primary and IHC probe
High
Background
Incubation time was too long
Reduce incubation time
Incubation temperature was too high
Reduce incubation temperature
Substrate incubation time was too long
Reduce substrate incubation time
Sections dried out
Avoid sections being dried out
Inadequate washing of sections
Wash at least 3 times between steps
Increase incubation with pre-treatment solution or 3% hydrogen
peroxide to block endogenous enzyme activities prior to incubation of
Tissue contains endogenous peroxidase antibody.
Non-specific binding of primary
Non-specific binding may be reduced by using higher dilution of
antibodies to tissue or antibody
primary antibodies or by using purified monoclonal or affinity-purified
concentration was too high
polyclonal primary antibodies.
Diffusion of tissue antigen due to
inadequate fixation
Increase duration of postfixation
Sections dried out
Avoid sections being dried out
Allied Biotech, Inc
10075 Tyler Place, Suite 19, Ijamsville, MD 21754
Tel: (301) 874-0495; Fax: (240) 465-5802
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