www.toyobo.co.jp/e/bio

F0992K

Instruction manual Can Get Signal immunostain 2004

Can Get SignalTM immunostain
Immunoreaction Enhancer Solution
NKB-401
NKB-501
NKB-601

5 mL x 2
20 mL
20 mL

Store at 4°C

Contents[1]

Introduction[2]

Components[3]

Protocol1. Materials required
2. Protocol for paraffin-embedded sections
3. Protocol for paraffin-embedded sections using secondary antibody reagents
previously optimized or for polymer complex method
4. Protocol for frozen sections
5. Protocol for cultured cells[4]

Reagent[5]

Troubleshooting

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory precaution and utilize safety while using this kit.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO （SHANHAI） BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
Can Get SignalTM immunostain is a reaction solution that contains an accelerator for
antigen-antibody reactions, which improves sensitivity, specificity, and S/N ofimmunohistochemistry (IHC) and immunocytochemistry.

Features-Can Get SignalTM immunostain improves sensitivity, specificity, and S/N of IHC.
-This system can be applied to various detection systems (e.g., chromogenic,chemiluminescence, or fluorescence).
-This system can be used with ABC or polymer complex methods.
-Can Get SignalTM immunostain consists of Solution A and B, which exhibit variousproperties for improving results. These reagents can be used independently.
-Reagents can be used directly without dilution. <Ready-to-use type>Fig. 1 Flow chart of ABC staining with IHC
Notes-Can Get SignalTM immunostain cannot be used as a blocking reagent. Blocking anddetection steps should be performed using conventional methods.
-This reagent is not applicable to the avidin-biotin reaction in ABC method[ 2 ] Components

This kit includes the following components. All reagents should be stored at 4°C andprotected from light.
Code No.
Reagent Name
NKB-401

NKB-501

NKB-601

Solution A

5 mL

20 mL

-Solution B

5 mL-20 mLNotes
Can Get SignalTM immunostain contains Solution A and B. These solutions exhibit
various acceleration effects, which are antigen/antibody-dependent, and can be used
independently. Both solutions should be examined prior to use.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio[3]Protocol

IHC is a method for detection of proteins located in tissue sections, which is accomplishedthrough the use of antibodies that recognize target proteins. The antibody-antigen
interaction is visualized by 1) chromogen detection, where an enzyme conjugated to an
antibody cleaves a substrate to produce colored precipitate at the protein location, or 2)
fluorescent detection, where a fluorophore is conjugated to an antibody and can bevisualized using fluorescence microscopy.
The following is a protocol for IHC in fixed tissue (typically neutral buffered formalin),which is embedded in paraffin prior to sectioning, using the ABC method with HRPconjugated antibodies. If secondary antibodies have been previously optimized, or for thepolymer complex method (e.g. ENVISION+, Dako), refer to [3] 3.

1. Materials required(1) Equipment:- Hellendahl jar- Slide- Coverslip- Mounting medium- Marker pen(2) Reagents and consumables:-Ethanol-Xylene-PBS*-Endogenous peroxidase blocking buffer*-Blocking reagent*-Chromogen substrate*See [7] Reagent

2. Protocol for paraffin-embedded sections(1) Place slides in rack and perform the following wash steps:-Xylene: three times for 3 minutes each wash-Ethanol: three times for 3 minutes each wash
-90% Ethanol: 3 minutes-80% Ethanol: 3 minutes-70% Ethanol: 3 minutes-Distilled water: 5 minutes-2 hours
Notes
Formalin-fixed tissue sections often require an antigen retrieval step prior to IHCstaining. During formalin fixation, methylene bridges between proteins are formed
and antigenic sites become masked. Several antigen retrieval methods are effective
for breaking the methylene bridges and exposing antigenic sites to allow antibodies
to bind. Heat-mediated (or heat-induced) or enzymatic antigen retrieval method is
generally sufficient.

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Tel(81)-6-6348-3888
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Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

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www.toyobo.co.jp/e/bio(2) Optional: Incubate in endogenous peroxidase blocking buffer for 30 minutes at room
temperature in the dark.
Notes
Some cells or tissues contain endogenous peroxidase. Endogenous peroxidase activity,which may cause high background, can be significantly reduced by pre-treating cells ortissues with hydrogen peroxide prior to incubation with HRP-conjugated antibodies.
(3) Wash in distilled water for 5 minutes, and two times in PBS for 5 minutes.
(4) Add 100 μL blocking reagent and incubate at room temperature for 1 hour.
(5) Dilute primary antibodies in Can Get SignalTM Immunostain Solution A or B to anappropriate concentration.
Notes
Can Get SignalTM Immunostain Solution A and B exhibit different acceleration effects,
depending on antigens and antibodies. These solutions can be used independently;
however, both solutions should be examined previously.
(6) After removing blocking reagent, add 100 μL diluted primary antibody solution andincubate at room temperature for 1 hour.
Notes
This reaction can be performed at 4°C overnight.
(7) Wash 3 times in PBS for 5 minutes.
(8) Dilute secondary antibodies in Can Get SignalTM Immunostain Solution A or B to an
appropriate concentration.
Notes-Can Get SignalTM Immunostain Solution A and B exhibit different acceleration
effects, depending on antigens and antibodies. These solutions can be usedindependently; however, both solutions should be examined previously.
-Optimal antibody concentrations tend to be lower in this method than conventional
methods. Therefore, antibody concentrations should be optimized based on lowerconcentrations.
(9) Subsequent to removal of primary antibody solution, add 100 μL diluted secondary
antibody solution, and incubate at room temperature for 1 hour.
(10) Wash 3 times in PBS for 5 minutes.

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www.toyobo.co.jp/e/bio(11) After removing residual PBS, add 100 μL avidin-biotin complex solution andincubate at room temperature for 30 minutes.
Notes
The avidin-biotin complex solution should be used within 30 minutes after preparation.
(12) Wash 3 times in PBS for 5 minutes.
(13) After removing residual PBS, add 200 μL substrate solution and incubate at room
temperature for an appropriate time.
(14) Rinse in distilled water to terminate the reaction.
(15) Optional: Counterstain.
(16) Mount coverslip with aqueous mounting medium or glycerol.

3. Protocol for paraffin-embedded sections using previously optimized
secondary antibody concentrations or the polymer complex method(1) Perform step (1)-(7) in [3] 2.
(2) After removing residual PBS, add 100 μL secondary antibody solution and incubate
at room temperature for 30 minutes.
(3) Perform step (10)-(16) in [3] 2.

4. Protocol for frozen sections(1) Wash the section 3 times in PBS for 10 minutes.
(2) Fix with the pre-cooled fixative (e.g., acetone) for 5-10 minutes at room temperature.
(3) Wash in PBS for 10 minutes.
(4) Perform (2)-(16) in [3] 2.
NOTES
The use of paraffin-embedded sections with previously optimized secondary antibody
concentrations, or the polymer complex method ([3] 3.), can be applied to thisprotocol.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo.co.jp/e/bio

5.

Protocol for cultured cells(1) Fix cells according to a general protocol.
(2) Optional: Permeabilize the cells using an appropriate detergent solution.
(3) Perform (2)-(16) in [3] 2.

[ 4 ] Reagents
1. 10X PBS(-) (10X PBS) (500 mL)
5.75 g Na2HP04•7H20
1.0 g
KH2PO440.0 g NaCl
1.0 g
KCl
Adjust volume to 500 mL

2. Endogenous peroxidase blocking solution (200 mL)
194 mL methanol
6 mL 10% H2O23. Blocking solution (10 mL)
10 mL 1X PBS(-)
150 μL normal serum
Notes
Animal species should be same between normal serum and secondary antibodies.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo.co.jp/e/bio[ 5 ] Troubleshooting
Symptom

Cause

Solution

Excessive primary antibody

Excessive secondary antibody
High background/Non-specific signalInsufficient blocking
Insufficient washing
Endogenous peroxidase
Excessive exposure time
(Fluorescent stain)
Insufficient primary antibody
Excessive blocking
Excessive washing
Lack of antigenicity
Weak signal
Masking of antigenicity
Antigen retrieval method isinappropriate
Excessive exposure time
(Fluorescent stain)

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

In this method, optimal concentrations tend to belower than conventional methods. Therefore,
antibody optimization should be based on thelower concentrations.
-In this method, the optimal concentrations forsecondary antibodies tend to be lower thanconventional methods. Therefore, antibody
optimization should be based on lower
concentrations.
-Previously optimized secondary antibodies canbe diluted with this reagent.
-Prolong blocking time.
-Change the blocking reagent.
Increase wash steps or time.
-Prolong treatment time with endogenous
peroxidase blocking buffer.
-Increase H2O2 concentration of endogenous
peroxidase blocking buffer up to 3%.
Decrease exposure time.
Increase concentration of primary antibodies.
- Optimize blocking time.
- Change the blocking reagent
Decrease wash steps or time.
-Tissue fixation method might be inappropriate.
Change fixation method.
-Antigen retrieval might be effective.
Formalin-fixed tissue sections often require
antigen retrieval prior to IHC staining.
Optimize antigen retrieval conditions.
Decrease exposure time. Excessive excitation
light bleaches fluorescence.

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

6