Catalog

ImmunohistochemistryAbout us
Nichirei Corporation had started bioscience business as a manufacture ofAnti-Leukocytes Monoclonal antibodies (CD Antibodies) in 1983.
Nichirei Biosciences Inc. (hereinafter called “Nichirei Bio” ) was reorganized on April 1,2005 succeeding Nichirei’ s bioscience business adopting the holding company systemby its parent company, Nichirei Corporation. Nichirei Bio’ s mission is to contribute to thedevelopment of the global Bio-industries including medical, cosmetic and healthcare
business as a specialized company by offering high-quality products and services
based on its advanced technology.

Nichirei Bio’s business field
1. IHC (Immunohistochemistory) products (-Histofine®)

Nichirei Bio has developed and improved patented state-of-the-art technology, UIP(Universal Immuo-enzyme Polymer) method. Nichirei Bio manufactures and supplies-Histofine® products including CE marked products adopted the UIP technology.
Also for domestic use, Nichirei Bio manufactures and supplies several IVD products
for IHC.

2. Diagnostic products (EIA and Lateral Flow Assay kits)
Nichirei Bio develops, manufactures and distributes several IVD products
immuno-reaction applied, especially POC (Point of Care) for rapid diagnostics.

3. Cell-biology products
Nichirei Bio provides animal sera and media related to cell culture to the customers
both in the academic and industrial field.

4. Functional Materials
Nichirei Bio develops functional materials (powder and extract) from natural raw
materials such as Acerola and Camu Camu fruits procured by consolidated
subsidiaries and affiliates of the Nichirei group. Nichirei Bio supplies such materials
to cosmetics and health food manufacturers.

1

Contents
About us………………………………………………………… 1

Contents………………………………………………………… 2

Highlights ………………………………………………………… 3-Histofine® series application on IHC Staining …………… 4

Products
Detection Systems for Tissue Sections
For Human Tissue Sections-Histofine® High Stain™ HRP (MULTI) ……………… 6-Histofine® Simple Stain™ MAX PO …………………… 8-Histofine® Simple Stain™ AP ………………………… 9-Histofine® ALK Detection Kit ………………………… 10
For Mouse・Rat Tissue Sections-Histofine® Simple Stain™ Rat MAX PO ……………… 12-Histofine® Simple Stain™ Mouse MAX PO ………… 13-Histofine® MOUSESTAIN KIT ………………………… 14
Enzyme Substrate Systems-Histofine® DAB-2V ……………………………………… 15
Staining Procedure forAdvantages of-Histofine® Simple Stain™ series …… 16-Histofine® Simple Stain™ series ………… 18

Technical Report
Technical Report 1 ………………………………………………20
Technical Report 2 ………………………………………………22
Technical Report 3 ………………………………………………25
Trouble Shooting………………………………………………… 26
References ……………………………………………………… 27
Product List ……………………………………………………… 28

2

Highlights
Two-Step Polymer Detection System-H-Histofine
Histofine® H
High
igh S
Stain™
tain™ H
HRP
RP ((MULTI)MULTI)-Histofine® High Stain™ HRP (MULTI) is the Two-Step Polymer Detection System for IHC
staining providing more amplified staining intensity compared with conventional One-steppolymer detection system.
This system is applicable to both of Mouse and Rabbit primary antibodies and is forformalin-fixed, paraffin-embedded tissue sections.
For
For details,
deta
ailss, seee PagePag
ge 6

DAB Substrate Kit-H-Histofine
Histofine® D
DAB-2V
AB-2
2V-Histofine® DAB-2V is used as chromogen-substrate reagents for peroxidase-based
immunohistochemical staining as well. DAB produces brown precipitates at the site of thetarget antigen or nucleic acid reacting with peroxidase.

Higher sensitivity and simplified preparation are available in comparison with prior-Histofine® DAB-3S kit.
For
For details,deetaaills, see
see Page
Page 15
5

Staining results with-Histofine® DAB-2V■ CD3 Rabbit-Monoclonal antibody(Code: 413591F)-Histofine® DAB-2V■ Actin, Smooth Muscle MouseMonoclonal antibody(Code: 412021F)

425312F

425314F

Components:

500 tests

1,500 tests

Reagent A: DAB solution concentrate
Reagent B: DAB buffer
Tube
Chip (For Reagent B)
Dropping bottle

1.2 ml x2
30 ml x2
x1
x1
x1

1.2 ml x6
30 ml x6
x1
x1
x1

3

-Histofine® series application on IHC staining

TissueEnzymeHRP

Primary antibody
Mouse orRabbit ab

High Stain™
HRP (MULTI)Mouse orRabbit ab

Simple Stain™
MAX PO (MULTI)Mouse ab

Simple Stain™
MAX PO (M)Rabbit ab

Simple Stain™
MAX PO (R)

Goat ab

Simple Stain™
MAX PO (G)Mouse orRabbit ab

Simple Stain™
AP (MULTI)Mouse ab

Simple Stain™
AP (M)

Rabbit ab

Simple Stain™
AP (R)

Rabbit ab

Simple Stain™
Mouse MAX PO (R)

Goat ab

Simple Stain™
Mouse MAX PO (G)Rat ab

Simple Stain™
Mouse MAX PO (Rat)

Mouse ab

MOUSESTAIN KIT

Mouse orRabbit ab

Simple Stain™
Rat MAX PO (MULTI)Mouse ab

Simple Stain™
Rat MAX PO (M)Rabbit ab

Simple Stain™
Rat MAX PO (R)

Goat ab

Simple Stain™
Rat MAX PO (G)Human Tissue

AP

Mouse Tissue

Rat Tissue4

Detection systems

HRP

HRP

Products

5Detection system for Human tissue sections-Histofine® High Stain™ HRP (MULTI)This product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine® High Stain™ HRP (MULTI) is the Two-Step Polymer Detection System for IHC staining providing
more amplified staining intensity compared with conventional One-step polymer detection system. This system isapplicable to both of Mouse and Rabbit primary antibodies and is for formalin-fixed, paraffin-embedded tissue
sections. It is the labeled polymer prepared by combining amino acid polymers with multiple molecules ofperoxidase (PO) and secondary antibody which is reduced to Fab’ fragment. To eliminate background staining,solid-phase adsorption of secondary antibody is conducted with human serum.

Principle & Procedure
Kit components
High Stain
Solution A

Step
Step 1High StainTM
Solution B

Step
Step 2

Add mouse or rabbit
primary antibody*.

TM

Add High StainTM
Solution A.

Fab' fragment of
secondary antibody

Human tissue
sectionPrimary antibody

Antigen
10 min.
Peroxidase

Amino-acidpolymer

Step
Step 4

Step
Step 3Add substratesolution*.

Add High StainTM
Solution B.

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10 min.

*Primary antibody and substrate solution are not provided in this kit.

Advantages
1. High intensity of staining.
2. Applicable to both of Mouse and Rabbit primary antibodies.
3. Low expression of antigen is detectable.
4. No background due to unaffected by endogenous biotin (Page 18).
5. Shortened reaction time.
6. Saving primary antibodies.

ProtocolAnƟbody

only for 20 minutes

High
igh StainTM
SoluƟon A
10 min6High StainTM
SoluƟon B
10 minDAB
chromogenCounterstaining

Comparison of Staining Results-Histofine®
High Stain™ HRP (MULTI)

Competitive Product■CDX-2 Rabbit-Monoclonal antibody (Code: 418011F)

Human colon cancer stained with -Histofine ® High Stain™ HRP (MULTI) and DAB chromogen.
Positive reaction is observed in nuclei of sporadic tumor cells.

■Cyclin D1 Rabbit-Monoclonal antibody (Clone: SP4) (Code: 413521F)

Human breast cancer (metastasis to liver) stained with -Histofine ® High Stain™ HRP (MULTI) and DAB
chromogen.
Positive reaction is observed in nuclei of sporadic tumor cells.

■ p40 Rabbit-Polyclonal antibody (Code: 418101F)

Human squamous cell cancer (lung) stained with -Histofine ® High Stain™ HRP (MULTI) and DAB
chromogen.
Positive reaction is observed in nuclei of tumor cells.

Product

Detection system.

Liquid. Ready to use.

Product Name

Tests

VolumeCode

For use with

Histofine® High Stain™ HRP (MULTI)

170

17ml each414481F

1,000

17ml x 6 each414483F

Mouse and Rabbit
primary antibodies-

High Stain™ Solution A
High Stain™ Solution B

7

Detection system for Human tissue sections-Histofine® Simple Stain™ MAX PO
This product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine® Simple Stain™ MAX PO is a detection reagent designed specifically to allow immunohistochemical
staining on formalin-fixed paraffin-embedded human tissue sections. It is the labeled polymer prepared by
combining amino acid polymers with multiple molecules of peroxidase (PO) and secondary antibody which is
reduced to Fab’ fragment. To eliminate background staining, solid-phase adsorption of secondary antibody isconducted with human serum.

Principle & Procedure
Fab' fragment of
secondary antibody-Histofine®

Simple Stain™ MAX PO
Peroxidase

Amino-acidpolymer

ng
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Human tissue
sectionAntigen
30 min.

Advantages

Add substrate solution.

Add polymer.

Add primary antibody.

*Page 18

1. Simplified staining steps*.
2. High sensitivity*.
3. No background due to unaffected by endogenous biotin*.
4. Ready to use.
■ Rabbit anti-S-100 protein
antibody■Mouse anti-Estrogen Receptor
antibody (clone: 1D5)

Human breast cancer (treated with hightemperature epitope unmasking method) stainedwith -Histofine® Simple Stain™ MAX PO(M) andDAB chromogen.
Nuclear staining of breast cancer cells is observed.

Human colon stained with-Histofine® Simple
Stain™ MAX PO(R) and DAB chromogen.
Cytoplasmic staining of nerve cells is observed.

Human breast cancer (treated with hightemperature epitope unmasking method) stainedwith -Histofine® Simple Stain™ MAX PO (MULTI)
and DAB chromogen.
Nuclear staining of breast cancer cells is observed.

Product

Detection systems.
Product Name-Histofine® Simple Stain™ MAX PO (MULTI)-Histofine® Simple Stain™ MAX PO (M)-Histofine® Simple Stain™ MAX PO (R)-Histofine® Simple Stain™ MAX PO (G)8Staining Results■ Mouse anti-Progesteron
Receptor antibody (clone:1A6)

Liquid. Ready to use.
For (slides)

Volume

Code

170
500
1500
170
500
1500
170
500
1500
170
500

17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3414151F
414152F
414154F
414131F
414132F
414134F
414141F
414142F
414144F
414161F
414162FFor use with
Mouse and rabbit
primary antibodies
Mouse
primary antibody
Rabbitprimary antibody
Goat
primary antibody

Mark in EU

Detection system for Human tissue sections-Histofine® Simple Stain™ AP
This product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine® Simple Stain™ AP is a detection reagent designed specifically to allow immunohistochemical staining
on formalin-fixed paraffin-embedded human tissue sections. It is the labeled polymer prepared by combining aminoacid polymer with alkaline phosphatase (AP) and secondary antibody which is reduced to Fab’ fragment. To
eliminate background staining, solid-phase adsorption of secondary antibody is conducted with human serum.

Principle & Procedure
Fab' fragment of
secondary antibody-Histofine®

Simple Stain™ AP
Alkaline
phosphatase

Amino-acidpolymer

ng
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Human tissue
sectionAntigen
30 min.

Add primary antibody.

Advantages

Add polymer.

Add substrate solution.

*Page 18

1. Simplified staining steps*.
2. High sensitivity*.
3. No background due to unaffected by endogenous biotin*.
4. Ready to use.

Staining Results■Mouse anti-Muscle Actin antibody(clone:HHF35)■Rabbit anti-S-100 protein antibody
Human stomach stained with -Histofine®
Simple Stain™ AP (M) and New fuchsin
chromogen.
Intense staining of smooth muscle in thewalls of blood vessel and muscularis
mucosae is observed.

Human colon stained with -Histofine®
Simple Stain™ AP (R) and New fuchsin
chromogen.
Cytoplasmic staining of nerve cells
scattered in smooth muscle and Auerbach’s
plexus is observed.

Product

Detection systems.

Liquid. Ready to use.

Product
ProductName
Name(slides)
ForTests

VolumeVolumeCode
Code

For use with-Histofine® Simple Stain™ AP (MULTI)

170
500
170
500
170
500

17ml x 1
17ml x 3
17ml x 1
17ml x 3
17ml x 1
17ml x 3414261F
414262F
414241F
414242F
414251F
414252F

Mouse and rabbit
primary antibodies-Histofine® Simple Stain™ AP (M)-Histofine® Simple Stain™ AP (R)Mouse
primary antibody
Rabbitprimary antibody

9Detection system for Human tissue sections-Histofine® ALK Detection KIT
For research use only

Feature-Histofine® ALK Detection Kit detects anaplastic lymphoma kinase (ALK) proteins in tumor cells inparaffin-embedded tissue specimens by IHC staining and determines presence of such protein expression.

Advantage
This kit is applicable to low expression of ALK fusion proteins as well.

Background
The ALK gene was identified in 1994 as a gene fused to the nucleophosmin (NPM) gene in anaplastic large-cell
lymphoma (ALCL) with t(2;5)(p23;q35) translocation. This gene is located at 2p23 and encodes for a receptor-type
tyrosine kinase, which belongs to the insulin receptor family. The ALK protein have a kinase domain in its intracellulardomain, and its function is associated with the promotion of cell growth and inhibition of apoptosis.
Subsequently, the ALK gene has been reported to form ALK fusion genes fused with ATIC, CLTC, MSN, TPM3, TPM4,
TFG, MYH9 and ALO17 genes in ALCL and also ATIC, CARS, CLTC, DCTN1, TPM3, TPM4, PPFIBP1, RANBP2 andSEC31L1 genes in inflammatory myofibroblastic tumor (IMT).
The proteins produced from these fusion genes are constantly activated by forming dimers and led to cancerous change.
Recently, other ALK fusion genes with EML4 gene, KIF5B gene or KCL1 gene in non-small cell carcinoma of lung,
SEC31A gene or SQSTM1 gene in ALK-positive large B-cell lymphoma and VCL gene in renal cell cancer have been
also reported.
■ Strong Positive Image■ Weak Positive Image

Lung adenocarcinoma: Positive reaction is observed in cytoplasm of tumor cells. Weak to strong positiveof staining levels is observed regarding the expression level of ALK fusion protein. Hot bath treatment (+)

Notes for Determination
Due to this kit detects ALK proteins, ALK fusion proteins as well as full-length ALK protein are reacted. Consequently,
results of slight positive to positive for tumors* that infrequently express full-length ALK protein are observed.
However, discrimination between ALK fusion proteins and full-length ALK protein is not available. Therefore,
considering expression possibility of ALK fusion proteins, confirmation of the presence or absence of ALK fusion
genes by using FISH method is preferable in this regard.
* Large-cell neuroendocrine carcinomas of the lung, small-cell lung carcinomas and rhabdomyosarcomas (particularly alveolar
rhabdomyosarcomas).

■ Weak positive Images in large cell neuroendocrine carcinoma of the lung

Weak positive staining is observed in the specimen which expresses full-length ALK protein.
Hot bath treatment (+)

10

Staining Flowchart
Note

Deparaffinization and Rehydration

High temperature epitope unmasking isrequired for formalin fixed paraffin-embedded
tissue sections prior to IHC staining.

Antigen Unmasking Treatment
Quenching of endogenous peroxidase
Reaction with Primary Antibody or Negative Control
Reaction with Bridge Reagent
Reaction with Peroxidase labeled Empower Reagent
Reaction with chromogen-substrate reagent
Counterstaining
Mounting

References(1) Shiota M., et al: Hyperphosphorylation of a novel 80 kDa protein-tyrosine
kinase similar to Ltk in a human Ki-1 lymphoma cell line, AMS3. Oncogene
9: 1567-1574, 1994
(2) Morris SW., et al: Fusion of a kinase gene, ALK, to a nucleolar protein
gene, NPM, in non-Hodgkin's lymphoma. Science. 263: 1281-1284, 1994
(3) Takeuchi K., et al: KIF5B-ALK, a novel fusion oncokinase identified by animmunohistochemistry-based diagnostic system for ALK-positive lungcancer. Clin Cancer Res. 15(9): 3143-3149, 2009(4) Takeuchi K., et al: Pulmonary inflammatory myofibroblastic tumor
expressing a novel fusion, PPFIBP1-ALK: reappraisal of anti-ALK
immunohistochemistry as a tool for novel ALK-fusion identification. Clin
Cancer Res. 17(10): 3341-3348, 2011(5) Sugawara E., et al: Identification of Anaplastic Lymphoma Kinase Fusions
in Renal Cancer. Cancer, 2012

-Histofine® ALK Control Slides
Both formalin fixed paraffin embedded cell lines of NCI-H2228 (Positive) and SK-BR-3 (Negative) are
mounted on each slide.
-Histofine® ALK Control Slides are used as a standard for discrimination of positive tissue blocks possess expressed
ALK protein.
This product is used for validation of reagent performance and staining technique in the IHC staining (including cytology
staining) with -Histofine® ALK Detection Kit.
Staining Images■ Negative control cell line/SK-BR-3 ■ Positive control cell line/NCI-H2228

Staining is not observed in the cytoplasm of all cells.
Treatment with hot bath (+)

Product

Detection system.

Liquid. Ready to use.
Product Name-Histofine® ALK D et e c t i o n K i t
Components
Vial No.
Blocking Reagent
1
Primary Antibody
2
Negative Control
3
Bridge Reagent
4
Peroxidase Labeled Empower Reagent
5
Chromogen Substrate
6
Substrate Buffer Solution
7
Chromogen Reagent
8
ALK Antigen Retrieval Solution A
9
ALK Antigen Retrieval Solution B
10

Product Name-Histofine®

Strong stainings are observed in the cytoplasm of
the majority of cells. Treatment with hot bath (+)

ALK Control Slides

Size

Code

20 tests

417071F

Constituents
3 V/V% Hydrogen peroxide
anti-ALK mouse monoclonal antibody (5A4)
Mouse IgG
3,3'-Diaminobenzidine tetrahydrochloride
0.6 V/V% Hydrogen peroxide solution

Size

Code

5 slides417081F

Volume
4ml × 1
2ml × 1
2ml × 1
4ml × 1
4ml × 1
0.5ml × 1
0.5ml × 1
0.5ml × 1
150ml × 1
150ml × 1

11

Detection system for Rat tissue sections-Histofine® Simple Stain™ Rat MAX PO
This product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine® Simple Stain™ Rat MAX PO is a detection reagent designed specifically to allow
immunohistochemical staining on formalin-fixed paraffin-embedded rat tissue sections. It is the labeled polymer
prepared by combining amino acid polymers with multiple molecules of peroxidase (PO) and secondary antibody
which is reduced to Fab’ fragment. To eliminate background staining, solid-phase adsorption of secondary
antibody is conducted with rat, human, dog, pig and bovine sera.

Principle & Procedure
Fab' fragment of
secondary antibody-Histofine®

Simple Stain™ Rat MAX PO
Peroxidase

Amino-acidpolymer

ng
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Rat tissue
sectionAntigen
30 min.

Add primary antibody.

Advantages

Add polymer.

Add substrate solution.

*Page 18

1. No reaction to endogenous rat immunoglobulin (Page 25).
2. Simplified staining steps*.
3. High sensitivity*.
4. No background due to unaffected by endogenous biotin*.
5. Ready to use.
■Mouse anti-Vimentin antibody (clone:V9)■Mouse anti-PCNA antibody (clone:PC10)■Goat anti-CD3-ε(M-20) antibody

Rat colon (treated with high temperature epitope
unmasking method) stained with-Histofine®
Simple Stain™ Rat MAX PO (M) and DAB
chromogen.
Cytoplasmic staining of fibroblasts and endothelial
cells is observed.

Rat liver stained with -Histofine® Simple Stain™
Rat MAX PO (MULTI) and DAB chromogen.
Nuclear staining of liver cells is observed.

Rat lymph node (treated with high temperature
epitope unmasking method) stained with-Histofine® Simple Stain™ Rat MAX PO (G)and DAB chromogen.
Membrane staining of almost all lymphocytes isobserved.

Product

Detection systems.
Product Name

12

Staining Results

Liquid. Ready to use.
For (slides)

VolumeCode

For use with-Histofine® Simple Stain™ Rat MAX PO (MULTI)

170

17ml

414191F

Mouse and rabbit
primary antibodies-Histofine® Simple Stain™ Rat MAX PO (M)

170

17ml

414171F

Mouse
primary antibody-Histofine® Simple Stain™ Rat MAX PO (R)

170

17ml

414181F

Rabbitprimary antibody-Histofine® Simple Stain™ Rat MAX PO (G)

170

17ml

414331F

Goat
primary antibody

Detection system for Mouse tissue sections-Histofine®Simple Stain™ Mouse MAX PO
This product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine® Simple Stain™ Mouse MAX PO is a detection reagent designed specifically to allow
immunohistochemical staining on formalin-fixed paraffin-embedded mouse tissue sections. It is the labeledpolymer prepared by combining amino acid polymers with multiple molecules of peroxidase (PO) and secondary
antibody which is reduced to Fab’ fragment. To eliminate background staining, solid-phase adsorption of
secondary antibody is conducted with mouse serum.

Principle & Procedure
Fab' fragment of
secondary antibody-Histofine®

Simple Stain™ Mouse MAX PO
Peroxidase

Amino-acidpolymer

ng
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Mouse tissue
sectionAntigen
30 min.

Add primary antibody.

Advantages

Add substrate solution.

Add polymer.

*Page 18

1. No reaction to endogenous mouse immunoglobulin (Page 25).
2. Simplified staining steps*.
3. High sensitivity*.
4. No background due to unaffected by endogenous biotin*.
5. Ready to use.
■Rabbit anti-Keratin/Cytokeratin
antibody

Mouse skin stained with-Histofine® Simple
Stain™ Mouse MAX PO(R) and DAB chromogen.
Cytoplasmic staining of epithelium cells and sweat
gland cells is observed.

Product

Staining Results■Goat anti-CD3-ε(M-20) antibody■Rat anti-Mouse CD45R/B220

Mouse lymph node (treated with high temperature
epitope unmasking method) stained with-Histofine® Simple Stain™ Mouse MAX PO(G)and DAB chromogen.
Membrane staining of almost all lymphocytes isobserved.

Mouse spleen stained with -Histofine® Simple
Stain™ Mouse MAX PO (Rat) and DAB chromogen.
Membrane staining of almost all lymphocytes ingerminal center and scattered interfollicularlymphocytes is observed.

Detection systems.
Product Name

Liquid. Ready to use.
For (slides)

Code

For use with

17ml

414341F

Rabbitprimary antibody

170

17ml

414351F

Goat
primary antibody

170

17ml

414311F

Rat
primary antibody

Histofine® Simple Stain™ Mouse MAX PO (R)-170-Histofine® Simple Stain™ Mouse MAX PO (G)-Histofine® Simple Stain™ Mouse MAX PO (Rat)

Volume13

Detection system for Mouse tissue sections-Histofine® MOUSESTAIN KITThis product adopted the UIP (Universal Immuno-enzyme Polymer) method, Nichirei Bio’s patented technology.

Feature-Histofine ® MOUSESTAIN KIT is the mouse on mouse system. It is designed specifically to allow
immunohistochemical staining with a mouse primary antibody on formalin-fixed paraffin-embedded mouse tissue
sections. This kit consists of Blocking reagent A, Blocking reagent B, and Simple Stain™ Mouse Max PO (M) which is thelabeled polymer prepared by combining amino acid polymer with multiple molecules of peroxidase and goat anti-mouse
Ig which is reduced to Fab’ fragment. To eliminate background staining, this kit uses Blocking reagent A and Blocking
reagent B.

Principle & Procedure
Fab' fragment of
secondary antibody

Endogenous
mouse Ig

Vial 1

Blocking
reagent A

Mouse tissue
sectionAntigenPeroxidase

Amino-acidpolymer

60 min.

Add Blocking reagent A.
Vial 3

Vial 2

Simple Stain™
Mouse MAX PO (M)Blocking
reagent B

10 min.

g
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10 min.

Add substrate solution.

Add polymer.

Add Blocking reagent B.

Advantages

Add primary antibody.

*Page 18

1. No reaction to endogenous mouse immunoglobulin.
2. Simplified staining steps*
3. High sensitivity*.
4. No background due to unaffected by endogenous biotin*.
5. Ready to use.

Staining Results■Mouse anti-PCNA antibody(clone:PC10)■Mouse anti-Muscle Actin antibody(clone:HHF35)
Mouse colon stained with -Histofine®
MOUSESTAIN KIT and DAB chromogen.
Nuclear staining of Epithelium cells isobserved.

Mouse colon stained with -Histofine®
MOUSESTAIN KIT and DAB chromogen.
Intense staining of smooth muscle andmuscularis propria in lamina propria
mucosae is observed.

Product

Detection system.
Product Name

14

Histofine®

MOUSESTAIN KIT

Liquid. Ready to use.
For (slides)

VolumeCode

For use with50

6ml x 3414321F

500

17ml x 9414322F

Mouse
primary antibody

Enzyme substrate for peroxidase-based IHC staining-Histofine® DAB-2V
Feature-Histofine® DAB-2V is used as chromogen-substrate reagents for peroxidase-based immunohistochemical
staining as well. DAB produces brown precipitates at the site of the target antigen or nucleic acid reacting withperoxidase.
Higher sensitivity and simplified preparation are available in comparison with prior-Histofine® DAB-3S kit.

Reagent preparation

Reagent A

Note
Mix carefully
without foaming.

Reagent B
Dropping bottle
Tube

Remove full dosage of the substrate solution from thetube to the dropping bottle using pipette.
Store in a dark place at 2 to 8 °C.
Use within two weeks.

Add 1 drop (approx. 20μl) of Reagent A per
500μl of Reagent B into graduated tube.

Procedure
Substrate
solution

Absorption
and wipe

Remove PBS or TBS
around tissue section.

at RT
for 5-20 min.

Wash the slides with tap water,counterstain and mount.

Apply prepared substrate solution
using dropping bottle coveringtissue section completely.

Advantages
1. Simple step for preparation of substrate solution.
2. Higher sensitivity than that of other DAB substrate kits.
3. Prepared substrate solution is available for two weeks.

Staining Results■Rabbit anti-CD3 antibody(clone:SP7)■Mouse anti-Smooth Muscle Actin(clone:1A4)

Human tonsil cancer
Positive reaction is observed in cytomembraneof sporadic tumor cells.

Human small intestine
Positive reaction is observed in cytoplasmof sporadic smooth muscle cells.

Product

Enzyme Substrate system
Product
ProductName
Name-Histofine® DAB-2V
Reagent A: DAB solution concentrate
Reagent B: DAB buffer
Tube
Chip (For Reagent B)
Dropping bottle

Liquid. Ready to use.
425312F Volume425314F
Tests
Code
500 tests

1,500 tests

1.2 ml x2
30 ml x2
x1
x1
x1

1.2 ml x6
30 ml x6x1
x1
x1

Mark in EU

515

Staining Procedure for--Histofine® Simple Stain™ Series-Histofine® Simple Stain™ MAX PO, Mouse MAX PO and Rat MAX PO● Deparaffinization and Rehydration (for paraffin-embedded tissue sections)xylene3 min.

xylene3 min.

xylene3 min.

100% ethanol 100% ethanol 95% ethanol 95% ethanol
3 min.
3 min.
3 min.
3 min.

PBS
5 min.

● Remove excess xylene and ethanol at each step.
● Change xylene and ethanol as appropriate to complete deparaffinization and rehydration.

● Quenching endogenous peroxidase
Absorbent
paper

Remove PBS around tissue sections.

Immerse the slides in 3 % of H2O2
solution in absolute methanol.
(at RT for 10-15 min.)

Rinse the slides in PBS.
(at RT for 5 min.)● Addition and reaction of primary antibody
Primary antibody
or negative control
Absorbent
paper

Remove PBS around tissue sections.

● Addition and reaction of

Apply primary antibody or negative
control (normal serum) to each slide
covering sections completely.
(at RT or 4°C )Rinse the slides in PBS.
(at RT for 5 min. x 3)

-Histofine® Simple Stain™ MAX PO, Mouse MAX PO or Rat MAX PO

Absorbent
paper

Remove PBS around tissue sections.

● Addition and reaction of

Absorbent
paper

Remove PBS around tissue sections.

-Histofine® Simple Stain™
*MAX PO, Mouse MAX PO,
or Rat MAX PO

Apply -Histofine® Simple Stain™* toeach slide covering sections completely.
(at RT for 30 min.)

Rinse the slides in PBS.
(at RT for 5 min. x 3)

-Histofine® Substrate Kit of Enzyme Substrate Systems*-Histofine® Simple Stain™
AEC Solution-Histofine® DAB-2V

Apply -Histofine ® Substrate Kit to eachslide covering sections completely.
(at RT for 5-20 min.)*User’s substrate solution is available as well.

Wash the slides with
distilled water.

● Counter staining● Mounting
As for alcohol soluble chromogens such as AEC, tissue sections are mounted with permanent mounting media, aqueous, without
further treatment. As for chromogens insoluble in organic solvent such as DAB, tissue sections are mounted with permanent
mounting media, non-aqueous, after washing with distilled water, dehydration in graded series of alcohol and clearing in xylene.

16● See instruction for use of primary antibody for further details such as quenching with endogenous peroxidase and hightemperature epitope unmasking methods.
● Use moist chamber for slide incubation.

-Histofine® Simple Stain™ AP● Deparaffinization and Rehydration (for paraffin-embedded tissue sections)xylene3 min.

xylene3 min.

xylene3 min.

100% ethanol 100% ethanol 95% ethanol 95% ethanol
3 min.
3 min.
3 min.
3 min.

PBS
5 min.

● Remove excess xylene and ethanol at each step.
● Change xylene and ethanol as appropriate to complete deparaffinization and rehydration.

● Addition and reaction of primary antibody
Primary antibody
or negative control
Absorbent
paper

Remove PBS around tissue sections.

● Addition and reaction of

Apply primary antibody or negative
control (normal serum) to each slide
covering sections completely.
(at RT or 4°C )Rinse the slides in PBS.
(at RT for 5 min. x 3)

-Histofine® Simple Stain™ AP-Histofine® Simple Stain™ AP

Absorbent
paper

Apply -Histofine® Simple Stain™
AP to each slide covering sections
completely. (at RT for 30 min.)Remove PBS aroundtissue sections.

● Addition and reaction of

Rinse the slide in PBS.
(at RT for 5 min. x 3)

Rinse the slides in TBS.
(at RT for 5 min. )-Histofine® Substrate Kit of Enzyme Substrate Systems*-Histofine® New Fuchsin
Substrate Kit**

Absorbent
paper

Remove PBS around tissue sections.

Apply-Histofine ® Substrate Kit to eachslide covering sections completely.
(at RT for 5-20 min.)*User’s substrate solution is available as well.

Wash the slides with
distilled water.

● Counter staining● Mounting
As for New Fuchsin substrate kit, tissue sections are mounted with permanent mounting media, aqueous, or air-dried,
cleared in xylene for a few seconds and mounted with permanent mounting media, non-aqueous.
● See instruction for use of primary antibody for further details such as quenching with endogenous peroxidase and hightemperature epitope unmasking methods.
● Use moist chamber for slide incubation.

**-Histofine® New Fuchsin Substrate Kit
A

B
Mix

distilled water
2mL

C
D

Mix

Mix
Use within 30 min.

Add 1drop of Reagent A
and 1drop of Reagent B

Add 2mL of
distilled water

Add 2drops of Reagent C

Add 1drop of Reagent D
and Mix

17

--Histofine® Simple Stain™ Series

Advantages of
Simplified staining steps(RT or 4°C)

(RT 30min.)

wa
M
ou
sh
in
nt
g
in
g

Conventional Streptavidin-biotin System*MAX PO, Mouse MAX PO,
Rat MAX PO and AP

ng

hi

s
wa

Protein Blocking

Primary antibody(RT 10min.)(RT or 4℃)

ng

hi

s
wa

ng

hi

s
wa

ng

hi

s
wa

M
ou
nt
in
g

-Histofine® Simple Stain™*

C
ou
nt
er
st
ai
ni
ng

Primary antibody

C
ou
nt
er
st
ai
ni
ng

hi
ng
wa
s

wa
s

hi
ng

-Histofine® Simple Stain™

Biotinylated
Streptavidin-enzyme
Secondary antibody conjugate(RT 10min.)(RT 5-10 min.)High sensitivity-Histofine® Simple Stain™
:-Histofine® Simple Stain™

: Biotin: Biotinylated secondary antibody: Streptavidin-enzyme conjugate

Primary antibody
Tissue section: Biotin

AntigenConventional Streptavidin-biotin System

Primary
ry antibody
Tissue section

Antigen

No background due to unaffected by endogenous biotin-Histofine® Simple Stain™

Conventional Streptavidin-biotin System

Backgro
Backgroundstaining

18

Technical Report19Technical Rep
Technical
Reportport 1

Application of--Histofine® MOUSESTAIN, Mouse MAX and Rat MAX
for mouse and rat frozen tissue sections-Histofine® MOUSESTAIN, Mouse MAX and Rat MAX for mouse and rat, paraffin embedded tissue sections, areapplicable for IHC staining with frozen tissue sections as well by following procedures.

Staining of frozen tissue sections with MOUSESTAIN KIT
MOUSESTAIN KIT is available for fixed frozen tissues without any change or addition on its procedure. (Principle & Procedure on page 14.)
1. Frozen tissue sections
Fixed frozen tissues* are only applicable.
*There are two different types of frozen tissues, Fresh frozen tissue and Fixed frozen tissue, used for IHC staining.
The fresh frozen tissue should be frozen immediately after the tissue obtained.
The fixed frozen tissue should firstly be fixed and then frozen.

2. Fixative solution
Apply fixative solution appropriate for primary antibody.
3. Concentration and reaction time of reagents
Apply equivalent concentration and reaction time of the respective reagents to these for paraffin embedded tissue sections.
In some preparation of frozen tissue sections, or regarding mouse lineages, tissues or fixing method, background staining may
be observed in this regard.
Staining Images
Fresh frozen tissue

Fixed frozen tissue
4% PFA (4°C, overnight)

Background staining in plasma cells
is observed.

Background staining in plasma cells
is not observed.

Detection system : MOUSESTAIN KIT
4% PFA (4°C, 10 min.)Tissue sections : mouse normal colon
Fixative solution : 4% of PFA (at 4 °C for overnight)
Primary antibody : PBS (in place of the primary antibody)
Chromogen: DAB

Staining of frozen tissue sections with-Histofinee® Mouse MAX
X and Rat MAX
X for mouse and rat tissue sections

Following Step A firstly and Step B-A secondly are recommended before the reaction with substrate solution to eliminate
background staining observed by the staining procedure. (Principle & Procedure on pages 12 and 13.)
1. Frozen tissue sections
Both fixed frozen tissues and fresh frozen tissues are applicable.
2. Fixative solution
Apply fixative solution appropriate for primary antibody.
Staining procedure-Histofine®
Simple Stain™ Mouse MAX
or Rat MAXmin
30 min.
Add primary antibody.

Add polymer.

Step B

20

ng
ni
i
a
g
st
er
tin
t
n
n
u
ou
M
Co

Add substrate solution.

Step AFirstly

Step Afor background staining observed by this staining procedure

Secondly

Step B

Step Afor background staining still observed by Step A

1.

Step A

Adjust reaction time of polymer.

30 min. of reaction time of polymer is designed for paraffin embedded sections.
For frozen tissue sections, apply adequate reaction time* reducing the duration between 10 to 30 min. when some backgroundstaining is observed by 30 min. reaction.
*The reaction time depends on mouse or rat lineages, tissues or fixing methods.
Step A-Histofine®
Simple Stain™ Mouse MAX
or Rat MAX

10 to 30 min.

g
in
in
ta
g
s
er
tin
nt
un
u
o
M
Co

Add polymer.

Add primary antibody.

Add substrate solution.

Staining Images
Background eliminated case by Step A

Reaction time of polymer: 30 min.

Reaction time of polymer: 10 min.
Step A

Detection system : Simple Stain™ Rat MAX PO (MULTI)
Tissue sections : rat normal colon
Fixative solution : 4% of PFA (at 4 °C for overnight)
Primary antibody : PBS (in place of primary antibody)
Chromogen: DAB

Background staining in plasma cells
is observed.

2.

Step B

Step A

Background staining in plasma cells
is not observed.

Add 0.2% of glutaraldehyde (GA) solution before the Step A.

Blocking with 0.2% of GA solution for 10 min.* may reduce background staining which is still observed by Step A.
Identify the absense of inhibition on the reaction of applied primary antibody prior to use of the GA solution.
*The effect of the blocking depends on mouse or rat lineages, tissues or fixing methods.
Dilution with SIGMA G7651 Dilute 250 times of SIGMA G7651, 50% concentration of GA, with PBS by 0.2% solution.
Others should be equivalent to above dilution.
Step B

Step A-Histofine®

0.2% GA solution
10 min.

Add primary antibody.

Add 0.2% of GA.

Simple Stain™
Mouse MAX
or Rat MAX

10 to 30 min..

Add polymer.

g
in
in
ta
g
s
er
tin
nt
un
u
o
M
Co
Add substrate solution.

Staining Images
Background eliminated case by Step B-A

Reaction time of polymer: 30 min.

Detection system : Simple Stain™
Mouse MAX PO (R)
Tissue sections : mouse normal colon
Fixative solution : 4% of PFA (at 4 °C for 10 min.)Primary antibody : PBS (in place of primary
antibody)
Chromogen: DAB
Background staining in plasma cells
is observed.

Reaction time of polymer: 10 min.

Reaction time of 0.2% GA: 10 min.
polymer: 10 min.

Step A

Step B

Background staining in plasma cells
is reduced but slightly observed.

Background staining in plasma cells
is not observed.

Step A

21

Technical Rep
Technical
Reportport 2IHC Triple-staining Method
The IHC triple-staining method with --Histofine® Simple Stain™ MAX PO (M),--Histofine
Histofinef ®SSimple S
Stain™ AP ((M)) and three differentffmurine primary antibodies
Stain™
I. OBJECTIVE
Detection of three different antigens at different locations
within the same tissue section
II. SPECIMENS
20% buffered formalin-fixed and paraffin-embedded tissue
section
III. TECHNICAL ADVICE (Staining orders)
1st Detection of Antigen:Detection of small amount of antigen with BCIP/NBT(Blue)
2nd Detection of Antigen:Detection of cytoplasmic antigen or large amount of
antigen with New Fuchsin (Red)
3rd Detection of Antigen:Detection of nuclear antigen or moderate to large amountof antigen with DAB (Brown)

IV. STAINING PROCEDURES
1. Deparaffinization and Rehydration
1-1. Immerse the slide in xylene at RT 3 times for 3 mineach.
1-2. Immerse the slide in 100% ethanol at RT 2 times for 3
min each.
1-3. Immerse the slide in 95% ethanol at RT 2 times for 3
min each.
1-4. Rinse the slide in PBS at RT for 5 min.
1st Detection of Antigen
2. Antigen Retrieval of the 1st primary antibody
2-1. Refer to the instruction for use of the 1st primary
antibodies and conduct proper antigen retrieval dependon the 1st primary antibody with specific buffer,specified temperature and incubation time, if necessary.
2-2. Allow the slide to cool down at RT for 20 - 60 min.
The slide should be cooled down slowly.
Rinse the slide in PBS at RT 3 times for 5 min each.
3. Protein Blocking
Apply 10% Goat normal serum at RT for 10 min.
4. Add 1st Primary Antibody
4-1. Apply 1st primary antibody at 37 °C for 1 hour.
4-2. Rinse the slide in PBS at RT 3 times for 5 min each.
5. Add -Histofine® Simple™ Stain AP (M)5-1. Apply -Histofine® Simple Stain™ AP (M) at RT for 30
min.
5-2. Rinse the slide in PBS at RT 3 times for 5 min each.
5-3. Rinse the slide in TBS at RT for 5 min.
6. Add BCIP/NBT substrate
6-1. Apply BCIP/NBT substrate solution.
Adjust the incubation time by microscopic observation.
6-2. Wash the slide with distilled water at RT for 5 min.

2nd Detection of Antigen
7. Antigen Retrieval of the 2nd primary antibody
7-1. Conduct Method-A or Method-B depend on the 2nd
primary antibody
Method-A for the 2nd primary antibody NO Antigen
Retrieval required
1) Fill the heat-resistant plastic staining jar with 10 mM
Sodium citrate buffer at pH 6.0 and heat to 95 °C.
2) Immerse the slide in the jar at 95 °C for 10 min*.
*Inactivation treatment for the 1st primary antibody and the
enzyme conjugated polymer of the 1st Detection of Antigen

Method-B for the 2nd primary antibody Antigen Retrieval
required
1) Fill the heat-resistant plastic staining jar with thespecific buffer referring the instruction for use of the
2nd primary antibody and heat to 95 °C.
2) Immerse the slide in the jar at 95 °C for 40 min**.
3) Allow the slide to cool down at RT for 20-60 min.
The slide should be cooled down slowly.
**Activation treatment for the 2nd primary antibody of the 2nd
Detection of Antigen, which is able to be combined with thetreatment of above Method A

7-2. Rinse the slide in PBS at RT 3 times for 5 min each.
8. Protein Blocking
Apply 10% Goat normal serum at RT for 10 min.
9. Add 2nd Primary Antibody
9-1. Apply 2nd primary antibody at 37 °C for 1 hour.
9-2. Rinse the slide in PBS at RT 3 times for 5 min each.
10. Add -Histofine® Simple Stain™ AP (M)10-1. Apply -Histofine® Simple Stain™ AP (M) at RT for 30
min.
10-2. Rinse the slide in PBS at RT 3 times for 5 min each.
10-3. Rinse the slide in TBS at RT for 5 min.
11. Add New Fuchsin substrate
11-1. Apply New Fuchsin substrate solution.
Adjust the reaction time by microscopic observation.
11-2. Wash the slide with distilled water at RT for 5 min.
3rd Detection of Antigen
12. Antigen Retrieval of the 3rd primary antibody
12-1. Conduct method-A or method-B depend on the 3rd
primary antibody
Method-A for the 3rd primary antibody NO Antigen
Retrieval required
1) Fill the heat-resistant plastic staining jar with 10 mM
Sodium citrate buffer at pH 6.0 and heat to 95 °C.
2) Immerse the slide in the jar at 95 °C for 10 min*.
*Inactivation treatment for the 2nd primary antibody and the
enzyme conjugated polymer of the 2nd Detection of Antigen

Method-B for the 3rd primary antibody Antigen Retrieval
required
1) Fill the heat-resistant plastic staining jar with thespecific buffer referring the instruction for use of the
3rd primary antibody and heat to 95 °C.
2) Immerse the slide in the jar at 95 °C for 40 min**.
3) Allow the slide to cool down at RT for 20-60 min.
The slide should be cooled down slowly.
**Activation treatment for the 3rd primary antibody of the 3rd
Detection of Antigen, which is able to be combined with thetreatment of above Method A

12-2. Rinse the slide in PBS at RT 3 times for 5 min each.
2213. Quenching of endogenous peroxidase
13-1. Immerse the slide in 3% H 2 O 2 solution in absolute
methanol at RT for 10 min.
13-2. Rinse the slide in PBS at RT 3 times for 5 min each.
14. Protein Blocking
Apply 10% Goat normal serum at RT for 10 min.
15. Add 3rd Primary Antibody
15-1. Apply 3rd primary antibody at 37 °C for 1 hour.
15-2. Rinse the slide in PBS at RT 3 times for 5 min each.
16. Add -Histofine® Simple Stain™ MAX PO (M)16-1. Apply -Histofine® Simple Stain™ MAX PO (M) at RTfor 30 min.
16-2. Rinse the slide in PBS at RT 3 times for 5 min each.
17. Add DAB substrate
17-1. Apply DAB substrate solution.
Adjust the reaction time by microscopic observation.
17-2. Wash the slide with distilled water at RT for 5 min.
18. Mounting
While the slide is wet by water, apply one drop of permanent
mounting media, aqueous, and fix with cover slip.
V. STAINING RESULTS
Case 1: Human Reactive Lymph Node

Case 2: Cervical Squamous Cell Carcinoma
1. OBJECTIVE
Observe three types of stained cells in a tissue section.
2. SPECIMENS
Cervical Squamous Cell Carcinoma
3. Used primary antibodies, antigen retrievals, detections andchromogens
1st Detection of Antigen:
Primary Antibody: Beta-catenin
Antigen Retrieval: 1mM buffered EDTA at pH8.0, 95 °C
for 40 min.
Detection system: -Histofine® Simple Stain™ AP (M)
Chromogen: BCIP/NBT (Blue)
2nd Detection of Antigen:
Primary Antibody: Cytokeratin (AE1/AE3)
Antigen Retrieval: 10mM Sodium citrate buffer at pH 6.0,
95 °C for 40 min.
Detection system: -Histofine® Simple Stain™ AP (M)
Chromogen: New Fuchsin (Red)
3rd Detection of Antigen:
Primary Antibody: Ki-67 antigen
Antigen Retrieval: 10mM Sodium citrate buffer at pH 6.0,
95 °C for 40 min.
Detection system: -Histofine® Simple Stain™ MAX PO (M)Chromogen: DAB (Brown)
4.Staining Images

1. OBJECTIVE
Observe three types of stained cells in a tissue section.
2. SPECIMENS
Human Reactive Lymph Node
3. Used primary antibodies, antigen retrievals, detections andchromogens
1st Detection of Antigen:
Primary Antibody: CD8
Antigen Retrieval: 1mM buffered EDTA at pH8.0, 95 °C
for 40 min.
Detection system: -Histofine® Simple Stain™ AP (M)
Chromogen: BCIP/NBT (Blue)
2nd Detection of Antigen:
Primary Antibody: CD4
Antigen Retrieval: 1mM buffered EDTA at pH8.0, 95 °C
for 40 min.
Detection system: -Histofine® Simple Stain™ AP (M)
Chromogen: New Fuchsin (Red)
3rd Detection of Antigen:
Primary Antibody: CD20cy
Antigen Retrieval: 10mM Sodium citrate buffer at pH 6.0,
95 °C for 40 min.
Detection system: -Histofine® Simple Stain™ MAX PO (M)Chromogen: DAB (Brown)

VI. ADVICE FOR STAINING
VI-1. Staining ScheduleTwo-day separated completion of all the steps of the IHC
triple-staining is available under the following conditions.
1st day
The reaction condition of 1st primary antibody should be
at 4 °C for overnight.
2nd day
The reaction condition of both 2nd and 3rd primary
antibodies should be at 37 °C for 1 hour.

4.Staining Images

523

VI-2. Chromogens Preparations
1. BCIP/NBT substrate solution
1-1. Reagents preparation
Substrate buffer (store at 2-8 °C):
100 mM Tris-HCl Buffer (100 mM sodium chloride, 50mM
MgCl2), pH 9.5
Adjust pH with HCl.
NBT stock solution (store at –20 °C):
Dissolve 75 mg of NBT (Nitro Blue Tetrazolium, SIGMA)in 1 ml of 70% N,N-dimethylformamid.
BCIP stock solution (store at –20 °C):
Dissolve 50 mg of BCIP (5-Bromo-4-Chloro-3-Indolyl
P h o s p h a t e - p - To l u i d i n e s a l t , S I G M A ) i n 1 m l o f
N,N-dimethylformamid.
1-2. Substrate solution preparation
Add 5 μl of BCIP stock solution and 6.5 μl of NBT stock
solution to 1.5 ml of Substrate buffer and mix well.
Use the solution within 30 min after preparation.
2. New Fuchsin substrate solution
2-1. Reagents preparation
Naphthol AS-BI phosphate solution (Use within 30 min
after preparation):
Dissolve 10 mg of naphthol AS-BI phosphoric acid
(SIGMA) in 100 μl of N,N-dimethylformamid.
New Fuchsin solution (store at 2-8 °C):
Dissolve 4.0 g of New Fuchsin powder (MERCK) in 100
ml of 2N HCl and filter the solution.
4 % Sodium Nitrite solution (Use within 30 min after
preparation):
Dissolve 40 mg of Sodium Nitrite in 1ml of distilled water
0.2 M Tris-HCl buffer (store at room temperature):
200mM Tris-HCl buffer, pH 8.2-8.3
Adjust pH with HCl.
2-2. Substrate solution preparation
Mix 100 μl of New Fuchsin solution and 100μl of 4 %Sodium Nitrite solution and incubate for 1 min.
Add 40ml of 0.2N Tris-HCl buffer to the mixture.
Add 100 μl of Naphthol AS-BI phosphate solution to the
mixture while stirring constantly.
Use the solution immediately after Filtration.
3. DAB substrate solution
Dissolve and mix following reagents and stir the solution.
Use the solution within 30 min after preparation.
10 mg of 3,3’-Diaminobenzidine, tetrahydrochloride
50 ml of 0.05 M Tris-HCl buffer, 15mM NaN3 pH 7.6
50 μl of 5 % H2O2 in distilled water
34 mg of Imidazole

24Technical Rep
Technical
Reportport 3

Advantages of Immunohistochemical polymer detection systems
designed for mouse & rat tissue sections
Introduction
When the immunohistochemical detection systems for human tissue sections are used for staining on mouse andrat tissue sections, background staining may be caused due to such reactivity with endogenous immunoglobulinsof mouse and rat in the tissue sections. Therefore, the immunohistochemical detection systems designed forstaining on mouse and rat tissue sections were developed. Background staining is compared among those
detection systems for mouse, rat and human tissue sections on mouse/rat tissue sections.

Methods

Materials & Methods
Materials
・Formalin-fixed paraffin-embedded mouse and rat tissue
sections
・ -Histofine® Simple Stain™ Mouse MAX PO (Rat) :rat primary antibody for mouse tissue sections
・ -Histofine® Simple Stain™ Rat MAX PO (M) :mouse primary antibody for rat tissue sections
・ -Histofine® Simple Stain™ MAX PO (M) :mouse primary antibody for human tissue sections

Steps ofimmunohistochemical staining

To compare background staining between polymers,
immunohistochemical staining on mouse tissue sections was
conducted with -Histofine® Simple Stain™ Mouse MAX PO(Rat) and -Histofine® Simple Stain™ MAX PO (M). (Fig.1)
To compare background staining between polymers,
immunohistochemical staining on rat tissue sections was
conducted with -Histofine® Simple Stain™ Rat MAX PO (M)and -Histofine® Simple Stain™ MAX PO (M). (Fig.2)
PBS was used in place of primary antibody to identify the
background staining caused by polymers.
DAB solution was used for brown color development.

Results

Quenching of endogenous peroxidase

It was found that the background staining by endogenous
immunoglobulins was not observed when the
immunohistochemical staining was conducted with thosepolymer detection systems designed for mouse and rat.

Fig.1 Staining comparison of Mouse tissue sections
Incubation with PBS for 30 min.

-Histofine®
Simple Stain™ Mouse MAX PO(Rat)
For Mouse tissue sections-Histofine®
Simple Stain™ MAX PO(M)For Human tissue sections

Incubation with polymer for 30 min.
Incubation with DAB solution for 10 min.

alrm
no loncoCounterstaining
Mounting
alrm
no leensp

Background staining is NOT observed. Background staining in plasma cells is observed.

Fig.2 Staining comparison of Rat tissue sections-Histofine®
Simple Stain™ Rat MAX PO(M)For Rat tissue sections-Histofine®
Simple Stain™ MAX PO(M)For Human tissue sections

alrm
no loncoBackground staining is NOT observed. Background staining in plasma cells is observed.

25

Trouble Shooting
ProblemPossible cause

Solution

1. Staining is not observed 1) Drying-out of tissue sections during ・Prevent tissue sections from drying out.
staining prior to addition of reagents.
or weak staining isobserved on positive
2) Embedding agent used is not suitable, ・Use suitable embedding agent or remove paraffin
control slide andor paraffin is not thoroughly removed thoroughly from tissue sections embedded.
specimen slide.
from paraffin-embedded tissue sections. ・Change xylene or ethanol in some cases.
3) Any trace amount of sodium azide ・Use sodium azide free buffer solution.
present
inbufferinactivates ・Change buffer solution.
peroxidase, such staining may not be
available.
4) Inadequate incubation of enzyme and ・Change stale chromogen-substrate reagent.
antibody.
・Remove excess solution thoroughly at each stage.
・Allow antibody sufficient time to react.
In particular, primary antibody should be incubated forthe specified time in its instruction for use.
1) Antigen is denatured or masked ・Some antigens are sensitive to fixation or embedding.
2. Specimen slide is not
Therefore use less potent fixative and shorten theduring fixing or embedding process.
stained while positive
fixing time.
control slide is stained.
・Heat-Induced Epitope Retrieval or treatment withproteolytic enzyme protease may be required forsome tissues to reveal antigens before staining.
2) Antigen is decomposed by autolysis. ・Use tissues obtained by biopsy or surgery.
3) Amount of antigen is few in tissue ・Prolong reaction time of reagent at each step.
sections.
3. Backgrounds are
Peroxidase staining
・Ensure treatment with 3% of hydrogen peroxide
intensively stained on
1) Endogenous enzyme activity was not added methanol to inactivate endogenous peroxidase
all the slides.
completely blocked.
activity.
・Before adding primary antibody, treat with 10%2) Non-specific staining is found.
normal goat or rabbit serum as follows.
Product nameSimple Stain™ MAX PO (M)
Simple Stain™ MAX PO (R)
Simple Stain™ MAX PO (MULTI)
Simple Stain™ MAX PO (G)Simple Stain™ Mouse MAX PO (R)
Simple Stain™ Mouse MAX PO (G)Simple Stain™ Mouse MAX PO (Rat)
Simple Stain™ Rat MAX PO (M)
Simple Stain™ Rat MAX PO (R)
Simple Stain™ Rat MAX PO (G)Simple Stain™ Rat MAX PO (MULTI)

Serum
goat
goat
goat
rabbitgoat
rabbit
goat
goat
goat
rabbitgoat

Alkaline phosphatase staining
・Add Levamisole to chromogen-substrate solution.
1) Endogenous enzyme activity was not To reduce endogenous enzyme activity,
completely blocked.
chromogen-substrate solution containing 1mM
Levamisole should be used.
・Before adding primary antibody, treat with 10%2) Non-specific staining is found.
normal goat serum as follows.
Product nameSimple Stain™ AP (M)
Simple Stain™ AP (R)
Simple Stain™ AP (MULTI)

Serum
goat
goat
goat

3) Autolysis results in excessive ・Use fresh tissues whenever available.
antigens isolated in histologicalsolutions.
・Change xylene or ethanol in some cases.
4) Insufficient removal of paraffin.
・Ensure thorough washing of antibody.
5) Insufficient washing of antibody.
6) High room temperature accelerates ・Keep room temperature at 15 to 25°C.
enzyme reactions.
・Shorten reaction time of enzyme.
7) Tissue sections are dried out during ・Prevent tissue sections from drying out.
staining after addition of reagents.
4. During reaction, tissue
sections come off fromslides.

261) Heat induced antigen retrieval ・Mount tissue sections on slides coated with anprocedure some antigens required or adhesive such as 0.02% poly-L-lysine or silane.
prolonged reaction time with primary
antibody may promote tissue sections
come off easier.

References
Human Tissue Sections(1) Kimura, N., et al: Synaptotagmin I Expression in Mast Cells of Normal Human Tissues, Systemic Mast Cell Disease, and a Human
Mast Cell Leukemia Cell Line. J. Histochem. Cytochem. 49: 341-346, 2001.
(2) Naito, Z., et al: Expression and accumulation of lumican protein in uterine cervical cancer cells at the periphery of cancer nests. Int. J.
Oncol. 20: 943-948, 2002.
(3) Hoshino, Y., et al: Maximal HIV-1 Replication in Alveolar Macrophages during Tuberculosis Requires both Lymphocyte Contact andCytokines. J. Exp. Med. 195: 495–505, 2002.
(4) Sawada, H., et al: Characterization of an Anti-Decorin Monoclonal Antibody, and Its Utility. J. Biochem. 132: 997–1002, 2002.
(5) Ozaki, K., et al: Mast Cell Tumors of the Gastrointestinal Tract in 39 Dogs. Vet. Pathol. 39:557-564, 2002.
(6) Zen, Y., et al: Lipopolysaccharide Induces Overexpression of MUC2 and MUC5AC in Cultured Biliary Epithelial Cells. Possible Key
Phenomenon of Hepatolithiasis. Am. J. Pthol. 161: 1475-1484, 2002.
(7) Sawada, H., et al: Case report: Altered decorin expression of systemic sclerosis by UVA1 (340–400 nm) phototherapy:
Immunohistochemical analysis of 3 cases. BMC Dermatol. 3:2, 2003.
(8) Takagi-Morishita, Y., et al: Mouse Uterine Epithelial Apoptosis is Associated with Expression of Mitochondrial Voltage-Dependent Anion
Channels, Release of Cytochrome c from Mitochondria, and the Ratio of Bax to Bcl-2 or Bcl-X1. Biol. Reprod. 68: 1178–1184, 2003.
(9) Morimoto, R., et al: Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-likemicrovesicles in rat pinealocytes. J. Neurochem. 84: 382-391, 2003.
(10) Nakatani, K., et al: Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis. Lab.
Invest. 84: 91-101, 2003.
(11) Kitada, M., et al: Translocation of Glomerular p47phox and p67phox by Protein Kinase C-beta Activation is Required for Oxidative
Stress in Diabetic Nephropathy. Diabetes. 52: 2603-2614, 2003.
(12) Eventov-Friedman S., et al: Embryonic pig liver, pancreas, and lung as a source for transplantation: Optimal organogenesis without
teratoma depends on distinct time windows. PNAS. 102: 2928-2933, 2005.
(13) Hsu W. M., et al: Calreticulin expression in neuroblastoma. a novel independent prognostic factor. Annals of Oncology. 16: 314-321,
2005.
(14) Sabourdy F., et al: Tumorigenic poxviruses: growth factors in a viral context? Journal of General Virology. 85: 3597-3606, 2004.
(15) Jacobi J., et al: Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury. Am J Physiol Heart Circ
Physiol. 290: 2051-2058, 2006.

Mouse・Rat Tissue Sections(1) Ezure, K., et al: Glycine Is Used as a Transmitter by Decrementing Expiratory Neurons of the Ventrolateral Medulla in the Rat. The
Journal of Neuroscience 23: 8941– 8948, 2003.
(2) Kitada, M., et al: Translocation of Glomerular p47phox and p67phox by Protein Kinase C-β Activation Is Required for Oxidative Stressin Diabetic Nephropathy. Diabetes 52: 2603–2614, 2003.
(3) Matsuyoshi, H., et al: Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing
Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination. The Journal of Immunology 172: 776-786, 2004.
(4) Noiri E., et al: Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol. 281: 948-957, 2001.
(5) Kazuyo, M., et al: Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. NATURE.
doi:10.1038/nature08636,2009.

ALK Detection KIT(1) Shiota M., et al: Hyperphosphorylation of a novel 80 kDa protein-tyrosine kinase similar to Ltk in a human Ki-1 lymphoma cell line,
AMS3. Oncogene 9: 1567-1574, 1994
(2) Morris SW., et al: Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science. 263:
1281-1284, 1994
(3) Takeuchi K., et al: KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for
ALK-positive lung cancer. Clin Cancer Res. 15(9): 3143-3149, 2009(4) Takeuchi K., et al: Pulmonary inflammatory myofibroblastic tumor expressing a novel fusion, PPFIBP1-ALK: reappraisal of anti-ALK
immunohistochemistry as a tool for novel ALK-fusion identification. Clin Cancer Res. 17(10): 3341-3348, 2011(5) Sugawara E., et al: Identification of Anaplastic Lymphoma Kinase Fusions in Renal Cancer. Cancer, 201227

Product List
For (slides)

Mark in EU

For Human Tissue Sections
170
1000

17ml ea. 414481F Mouse and rabbit
17ml x 6 ea. 414483F primary antibodies

170
500
1500
170
500
1500
170
500
1500
170
500
170
500
170
500
170
500

17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3
17ml x 9
17ml x 1
17ml x 3414151F
Mouse and rabbit
414152F
primary antibodies
414154F
414131F
Mouse
414132F primary antibody
414134F
414141F
Rabbit414142F
primary antibody
414144F
414161F Goat
414162F primary antibody

17ml x 1
17ml x 3
17ml x 1
17ml x 3
17ml x 1
17ml x 3414261F Mouse and rabbit
414262F primary antibodies
414241F Mouse
414242F primary antibody
414251F Rabbit
414252F primary antibody-Histofine® Simple Stain™ Mouse MAX PO (R)170

17ml x 1

Rabbit414341F primary antibody-Histofine® Simple Stain™ Mouse MAX PO (G)170

17ml x 1

Goat414351F primary antibody-Histofine® Simple Stain™ Mouse MAX PO (Rat)

170

17ml x 1

Rat
414311F primary antibody-Histofine® MOUSESTAIN KIT

50500

6ml ea. 414321F Mouse
17ml x 3 ea. 414322F primary antibody-Histofine® Simple Stain™ Rat MAX PO (MULTI)

170

17ml x 1

and rabbit
414191F Mouseprimary antibodies-Histofine® Simple Stain™ Rat MAX PO (M)170

17ml x 1

Mouse
414171F primary antibody-Histofine® Simple Stain™ Rat MAX PO (R)170

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Rabbit414181F primary antibody-Histofine® Simple Stain™ Rat MAX PO (G)170

17ml x 1

Goat414331F primary antibody-Histofine® High Stain™ HRP (MULTI)High Stain™ Solution A
High Stain™ Solution B-Histofine® Simple Stain™ MAX PO (MULTI)-Histofine® Simple Stain™ MAX PO (M)-Histofine® Simple Stain™ MAX PO (R)-Histofine® Simple Stain™ MAX PO (G)-Histofine® Simple Stain™ AP (MULTI)-Histofine® Simple Stain™ AP (M)-Histofine® Simple Stain™ AP (R)For Mouse Tissue Sections

Blocking Reagent A
Blocking Reagent B
Simple Stain™ Mouse Max PO (M)For Rat Tissue Sections

Enzyme Substrate Systems-Histofine® DAB-2V
Reagent A : DAB solution concentrate*
Reagent B : DAB buffer**-Histofine® DAB-3S kit

500
1500
500
1500

*1.2ml x 2**30ml x 2*1.2ml x 6**30ml x 6
3ml ea.
9ml ea.

425312F
Peroxidase
425314F
415192F
Peroxidase
415194F

Reagent A : DAB solution concentrate
Reagent B : Enhancer solution concentrate
Reagent C : Hydrogen peroxide solution concentrate-Histofine® Simple Stain™ AEC Solution

500
1500

-Histofine® New Fuchsin Substrate kit

2000

Reagent A : New Fuchsin solution concentrate*
Reagent B : Activating reagent concentrate*
Reagent C : Substrate buffer concentrate**
Reagent D : Substrate solution concentrate*

28

17ml x 3 415182F
Peroxidase
17ml x 9 415184F* 6ml ea.
Alkaline** 12ml x 1 415161F phosphataseLiquid. Ready to use.
Size
For Human Tissue Sections-Histofine® ALK Detection Kit
1
2
3
4
5
6
7
8
9
10

4ml × 1
2ml × 1
2ml × 1
4ml × 1
4ml × 1
0.5ml × 1
0.5ml × 1
0.5ml × 1
150ml × 1
150ml × 1

Blocking Reagent
Primary Antibody
Negative Control
Bridge Reagent
Peroxidase Labeled Empower Reagent
Chromogen Substrate
Substrate Buffer Solution
Chromogen Reagent
ALK Antigen Retrieval Solution A
ALK Antigen Retrieval Solution B-Histofine® ALK Control Slides

417071F

20 tests

5 slides417081F

Storage
Store in a dark place at 2 to 8 °C.

29

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2016.10