Can Get Signal ® immunostain
Immunoreaction Enhancer Solution(Code No. NKB-401, NKB-501, NKB-502, NKB-601, NKB-602)

Instruction Manual

TOYOBO CO., LTD. Life Science Department
OSAKA JAPANA3327K-0-

− Contents −[1] Introduction・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 1[2] Composition ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 2[3] How to use ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 3[4] How to stain paraffin-embedded section ・・・・・・・・・・・・・・・・・・・・・・・・・ 4[5] How to stain frozen sections ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 6[6] How to prepare reagents ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 7[7] Trouble shooting

・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 8[8] Related products ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 9

CautionAll reagents contained in this kit are for research purposes only. Never use them asreagents for diagnosis or other clinical purposes. Comply with general laboratory
precautions and rules and ensure safety when using this kit.

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[1] Introduction
Can Get Signal® immunostain has been developed as a reaction-enhancing
kit to address the problems often encountered during immunohistological staining
and immunostaining of cells. These include inadequate sensitivity, low S/N ratio, andthe appearance of nonspecific signals from secondary antibodies. In the past,
inadequate detection sensitivity due to low antibody titers was dealt with byincreasing the concentration of the added antibody. However, increasing theantibody concentration often caused background and nonspecific responses. Byenhancing antigen-antibody reactions using this kit, it will improve the ability to
optimize the antibody concentration, lead to clearer staining responses while
suppressing nonspecific reactions caused by excessive addition of antibodies.

◆ Features ◆1: Enhances immunostaining signal
Can Get Signal® immunostain enhances antigen-antibody reactions,thus yielding higher signal compared with conventional techniques. With thiskit it is possible to obtain images with a high S/N ratio and suppressedbackground signals for the detection of fluorescence or other light by reducing
the amount of secondary antibody used or shortening the exposure time.
2: Applicable to a range of antibodies
Can Get Signal® immunostain contains two solutions with different
effects on antibodies (Solution A and Solution B). The reagent most suitable toa given experimental system can be selected. (When you first use this kit, it isrecommended to use the Starter Set composed of both solutions, as describedon the next page).
3: Applicable to a range of systems
None of the components of Can Get Signal® immunostain affect labelingenzymes or fluorescent dyes. The kit is therefore applicable to systems for
detection of chemical coloration, chemiluminescence, and fluorescence. The
kit can also be used in combination with sensitizing systems such as the ABC orpolymer complex method.
4: User-friendly ready-to-use
Can Get Signal® immunostain can be used without prior dilution.

-2-[2] Composition

This product is made up of the following components.

Product and composition®

Can Get Signal immunostain

Package

Code No.

Each 5 ml

NKB-401

20 ml

NKB-501

20 ml x4

NKB-502

20 ml

NKB-601

20 ml x4

NKB-602

Starter Set

Solution A
Solution B
Can Get Signal ® immunostain
Solution A®

Can Get Signal immunostain
Solution B

All reagents need to be stored protected from light at 4°C.

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[3] How to use(1) Antibody (primary antibody or labeled secondary antibody) is diluted with this kit
and it is used directly for the assay. This kit is usually used to replace TBS(TBS-T), PBS (PBS-T) or blocking buffers for dilution of antibodies.
(2) This kit enhances antigen-antibody reactions, resulting in stronger signals thanwith conventional techniques. Due to an increase in reactivity, higher
background signals may also occur. In such cases, the antibody concentration
is probably not optimal, and it will be difficult to obtain optimal staining
responses using the same conditions as conventional techniques. To
resolve this problem, it is advisable to review the concentration of the
primary or secondary antibody used for detection or to readjust the signal
detection time (exposure time) and other parameters.
(3) There are two types of reagents (Solution A and Solution B) (see Previous
Page for the composition of the kits). Solution A and Solution B have different
effects on antibodies. The most suitable solution depends on the type ofantibody, antigen, and sample involved. It is advisable to try both solutions
for a given assay system and select the solution that gives the best results.
A Starter Set composed of both Solution A and Solution B is available to selectthe more suitable of the two solutions (see Previous Page).
* Precautions *• If a labeled secondary antibody or a polymer complex reagent is prepared inadvance to an appropriate dilution for detection, this kit should be used only fordilution of the primary antibody. However, if the secondary antibody is to bediluted before use, this kit can be used as a diluent.
• This kit cannot be used for any step, such as the blocking reaction or avidin-biotin
complex formation of ABC method, other than antigen-antibody reactions. This kit
does not enhance the former reactions and, in some cases, the kit adversely
affects them.

-4-[4] How to stain paraffin sections
A method of peroxidase immunostaining using the ABC technique is presented
below for reference. Various other immunostaining techniques are available,
including methods using other enzymes or fluorescent dyes for labeling and methods
using tyramide-based sensitization systems. Can Get Signal® immunostain isapplicable to all such methods. If you plan to use secondary antibodies prepared inadvance at an appropriate dilution or to use reagents for the polymer complex
technique such as ENVISION+ (DAKO) and Histofine Simple Stain (Nichirei
Corporation), you should also read (4) Staining with a secondary antibody
prepared in advance at an appropriate dilution.
(1) Materials needed• Staining bottle (Doze)• Humidifying box• Glass slide• Marker pen for
immunohistological staining• Glass coverslip• Timer• Mounting agent(2) Reagents needed(*See [6] How to prepare reagents)• Ethanol• Xylene• Endogenous peroxidase
inactivating fluid*• PBS*• Blocking fluid*• Substrate solution for color
development (TMB, DAB, etc.)(3) Staining procedure
1. Deparaffinize section in 2 changes of 100 % xylene for 5 min each, followed byhydration with ethanol.
2. Wash section with distilled water for at least 5 min.
3. If endogenous peroxidase inactivation is necessary, expose section to an endogenous
peroxidase inactivating fluid for 10 min.
4. Wash section with distilled water for 5 min, followed by two changes of PBS, 5 mineach.
5. Drop 100 µl of the blocking fluid onto the section and leave standing for 60 min toeffect blocking.

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6.

After removing excess blocking fluid from the section, drop a solution of primary
antibody, pre-diluted with Solution A or B of Can Get Signal® immunostain, onto thesection and leave standing for 60 min.
・ See [3] How to Use for details about the selection of Solution A or B and the antibody dilution ratio.
・ This reagent can also be used in combination with blocking agents such as BSA, casein or normal
serum. Before diluting the antibody, these blocking agents need to be combined with this reagent.

7. Wash section with 3 changes of PBS, 5 min each.
8. After removing excess PBS from the section, drop a solution of secondary antibody,pre-diluted with Solution A or B of Can Get Signal® immunostain, onto the section
and leave standing for 60 min.
・ See [3] How to Use for details about the selection of Solution A or B and the antibody dilution ratio.
・ This reagent can also be used in combination with blocking agents such as BSA, casein or normal
serum. Before diluting the antibody, these blocking agents need to be combined with this reagent.

9. Wash section with 3 changes of PBS, 5 min each.
10. After removing excess PBS from the section, drop 100 µl of an avidin-biotin complex
solution onto the section and leave standing for 30 min.

Prepare the avidin-biotin

complex solution 30 min prior to use.
11. Wash section with 3 changes of PBS, 5 min each.
12. After removing excess PBS from the section, drop about 200 µl of a substrate solution
onto the section to develop colors. Since the time required for color development
varies greatly depending on the experimental system, the reaction needs to bemonitored under a microscope and stopped at an appropriate time.
13. Immerse section in distilled water to stop reactions.
14. Mount section with glycerin or mounting agents and observe. Counterstain as needed.
Dehydrate with ethanol and xylene and mount for observation.
The appropriate mounting agent depends on the color developer used. Read the instructions
carefully for both the color developer and the mounting agent you use.

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(4) Staining with the secondary antibody prepared in advance at an appropriate
dilution
If you plan to use secondary antibodies prepared in advance at an
appropriate dilution, or to use reagents for the polymer complex technique such as
ENVISION+ (DAKO Cytomation) and Histofine Simple Stain (Nichirei Corporation),
you can only use this reagent for diluting the primary antibody.
1. Follow steps 1 through 7 in (3) Staining procedure.
2. After removing excess PBS from the section, drop 100 µl of the secondary
antibody solution onto the section and leave standing for 30 min.
You need to follow the instructions regarding the dilution, temperature, and time for the reagents used.
If you plan to dilute the secondary antibody, you may use Solution A or B of Can Get Signal®
immunostain as a diluent.

3. Follow step 9 and subsequent steps in (3) Staining procedure.
You need to follow the instructions regarding washing and color development for each reagent used inthese steps.

[5] How to stain frozen sections
1. Wash section with 3 changes of PBS, 10 min each.
2. Immediately before staining, fix the section using the method appropriate forthe antigen used (such as acetone fixation).
3. Wash section with PBS for 10 min.
4. If endogenous peroxidase inactivation is required, follow Step 3 of How tostain paraffin-embedded sections.
5. Wash section with 3 changes of PBS, 5 min each.
6. Follow step 5 and subsequent steps in [4] How to stain paraffin-embedded
sections.

-7-[6] How to prepare reagents・10x PBS(-) (10x PBS) (500 ml)
43 mM Na2HPO4・7H2O (MW = 268.07)
14 mM KH2PO4 (MW = 136.09)
27 mM KCl (MW = 74.55)
1.37 M NaCl (MW = 58.44)
Dissolve Na2HPO4 ・7H2O (5.75 g), KH2PO4 (1.0 g), NaCl (40.0 g) and KCl(1.0 g) in distilled water, to give a 10x solution (500 ml). Autoclave the solution
as needed. The solution can be stored refrigerated or at room temperature.
If 10x PBS is refrigerated, sedimentation may occur due to the high salt
concentration. If sediment forms, warm the PBS in a water bath (37°C), tocompletely dissolve the sediments before use.
Prepare 1x PBS by 1:10 dilution of 10x PBS with distilled water or sterile
distilled water. Store the 1x PBS refrigerated or at room temperature.
• Endogenous peroxidase inactivating fluid (0.3% hydrogen peroxide/methanol; 200ml)
Add 10% Hydrogen peroxide (6 ml) to methanol (194 ml), and agitate to mix
thoroughly. Prepare this fluid immediately before use. Do not add the methanol
to the hydrogen peroxide. Generation of intense heat may occur.
• Blocking solution (1.5% normal serum/PBS; 10 ml)
Add 150 µl normal goat serum to 10 ml of 1x PBS(-).
If normal serum is used as a blocking agent, the serum is usually collected fromanimals of the same species as those from which the secondary antibody isderived.

-8-[7] Trouble shooting
Problem

Cause

CountermeasureHighbackground or
nonspecificstaining
responses

The use of this reagent
has reduced the optimal
concentration of the
primary antibody.

Reduce the concentration of the primary
antibody

The concentration of thesecondary antibody is too
high.

Reduce the concentration of thesecondary antibody. If you plan to use asecondary antibody prepared inadvance at an appropriate dilution,
dilute it with this reagent.

Conditions for blocking
are inappropriate

Review the blocking time as the lengthof blocking time can greatly affect thestaining response. In some cases,changing the type of blocking agent may
lead to favorable results.

Washing is insufficient.

Increase the duration and frequency of
washing.

Endogenous peroxidase
activity remains (ifperoxidase-labeled
antibody is used).

Extend the duration of the inactivating
step or reduce the concentration ofhydrogen peroxide to 3%.

Exposure time is too long(for fluorescent staining)

Shorten the exposure time.

The concentration of the
primary antibody is low.

Increase the antibody concentration.

Blocking is excessive.

Review the blocking time as the lengthof blocking time can greatly affect thestaining responses. In some cases,changing the type of blocking agent may
lead to favorable results.

Washing is excessive.

Reduce the duration and frequency of
washing.

Antigenicity has beenlost.

Change the fixation method, as the
problem may be associated with the
fixation method used. In some cases,
antigen potentiation may lead tofavorable results.

Antigen has beenmasked.

Unmask the antigen. Depending on thenature of the sample tested, theproblem may be associated with theinability of the antibody to physicallybind the antigen.

Weak signal-9-

Low stability ofthe mounted
sampleUnmasking is
inappropriate.

Review the unmasking conditions, since
excessive unmasking can lead to loss ofantigenicity.

Exposure time is too long
(fluorescent staining).

Shorten the exposure time, since toolong an exposure to excitation can
bleach the fluorescent dye.

The mounting agent is
inappropriate.

Read the instructions for the color
developer, fluorescent dye, and themounting agent carefully. The
appropriate mounting agent can varydepending on the color developer used
and because the normal mounting
agents for fluorescent dyes are notapplicable to Alexa Fluor series
fluorescent dyes.

[8] Related products
Product and composition
Can Get Signal ®
Immunoreaction Enhancer Solution(For Western blot and ELISA)Standard size (solutions for primary andsecondary antibodies)
Trial size (solutions for primary andsecondary antibodies)
Solution for primary antibody only
Solution for secondary antibody only

Package

Storage

Code No.

Price

Each 250 ml

4°C

NKB-101¥30,000

Each 50 ml

4°C

NKB-101T¥10,000

250 ml
250 ml4°C
4°C

NKB-201
NKB-301¥17,000¥17,000◆In addition, Toyobo sells the products of the following antibody manufacturers.
See our on-line catalogue or each manufacturer’s website for details.
On-line Catalogue, Life Science Division, Toyobo, Ltd.: www.bio.toyobo.co.jp/bio01
・ Santa Cruz Biotechnology, Inc. www.scbt.com
A wide range of antibodies, with emphasis on proteins involved in signal transduction. Reagents andkits for immunoassay are also available.

・ PeproTech, Inc.

www.peprotech.com

Antibodies to growth factors and cytokines are available.

・ Fitzgerald Industries International, Inc. www.fitzgerald-fii.com
A wide range of antibodies is available including not only antibodies to cytokines and those involved insignal transduction but also antibodies to viruses, hormones, and narcotics.

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