For research use only

September 2011

-Histofine®
ALK Detection Kit

Package: 20 tests
Storage: Store at 2-8°C

PRECAUTIONSSTAINING PROCEDURE

1. Read these instructions carefully before use.
2. Handle specimens with care in view of risks of infection.

1. Staining Flowchart
Deparaffinization and rehydration

KIT COMPONENTS↓
Reagents

Vial No.

Code: 417071F

Antigen unmasking treatment

Volume↓Quenching of endogenous peroxidase↓1

Blocking Reagent
3 V/V% Hydrogen peroxide

4 ml ×1

2

Primary Antibody
anti-ALK mouse monoclonal antibody
clone: 5A4

2 ml ×1

Reaction with Bridge Reagent

3

Negative Control
Mouse IgG

2 ml ×1

Reaction with Peroxidase Labeled Empower Reagent

4

Bridge Reagent

4 ml ×1

Reaction with Chromogen/Substrate Reagent

5

Peroxidase Labeled Empower Reagent

4 ml ×1

6

Chromogen Substrate
3,3'-Diaminobenzidine tetrahydrochloride

0.5 ml ×1

7

Substrate Buffer Solution

0.5 ml ×1

8Chromogen Reagent
0.6 V/V% Hydrogen peroxide solution

0.5 ml ×1

9

ALK Antigen Retrieval Solution A

150 ml ×1

10

ALK Antigen Retrieval Solution B

150 ml ×1

Reaction with Primary Antibody or Negative Control↓↓↓↓

Counter staining↓

Mounting
2. Specimen Preparation

For optimal fixation to maintain tissue morphology and antigen activity,
it is recommended to use tissue sections as fresh and as small (approx. 1
cm × 1 cm × 0.5 cm) as possible, and the fixatives shown below:
Fixative
10% formalin or buffered formalin
20% formalin

INTENDED USEThis kit uses immunohistochemical (IHC) staining to detect anaplastic
lymphoma kinase (ALK) proteins in tumor cells, and to determine thepresence or absence of expression of these proteins.

Fixing duration
24–48 hours
12–24 hours

3. Preparation of Sections and Slides(1) Paraffin-Embedded Tissue Sections
Slice sections into 4-µm thickness and place on poly-L-lysine orsilane-coated slides. Place in incubator at 37°C and dry for 24hours.

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.(1)(2) This gene is located at 2p23 andencodes for a receptor-type tyrosine kinase, which belongs to the insulin
receptor family. The ALK protein have a kinase domain in its
intracellular domain, and its function is associated with the promotion ofcell growth and inhibition of apoptosis.
In ALCL and inflammatory myofibroblastic tumor, the ALK gene has
been reported to fuse with genes such as TFG, ATIC, CARS, CLTC,
SEC31L1, RANBP2, and TPM3, to form ALK fusion genes. The proteinsthat are produced from these ALK fusion genes form dimers, and as aresult, the kinase domain of the ALK gene was constitutively activated,leading to carcinogenesis.

(2) Specimen Sample Slides
Prepare 2 slides per specimen as specimen sample slides.
Use one as the Reagent Control slide in the staining process with
the Negative Control, Mouse IgG, instead of the Primary Antibody.
(3) Specimen Control Slides
a. Positive Control Slide
Slide is prepared with the same method as the specimen sample
slides; presence of ALK protein overexpression is confirmed inadvance.
b. Negative Control Slide
Slide is prepared with the same method as the specimen sample
slides; absence of ALK protein overexpression is confirmed inadvance.

Recently, fusion of the following genes with the ALK gene has beenreported: the EML4 and KIF5B genes in non-small cell lung carcinoma,
the SEC31A and SQSTM1 genes in ALK-positive large B-cell lymphoma,
and the VCL gene in renal cell carcinoma.

Conduct the entire process, from specimen preparation to staining andmicroscopic observation, after preparing and reviewing specimen
control slides, specimen sample slides, and reagent control slides.

PRINCIPLE

4. Procedure

ALK Detection Kit is composed of reagents for the detection of ALK
proteins. The antigen on formalin-fixed paraffin-embedded tissue
sections is reacted with Primary Antibody, then reacted with Bridge
Reagent. In addition, it is reacted with Peroxidase-Labeled Empowered
Reagent. This results in the formation of an antigen・antibody・enzyme
complex. Color development is observed when the enzyme in the complex
reacts with the substrate. This allows highly sensitive visualization of
antigen sites of and reliable confirmation of the presence of antigens byusing a light microscope.

(1) Reagents and Materials required but not provided· Slide glass (poly-L-lysine or silane-coated)· Drying apparatus· Timer· Staining racks or Coplin jars
· Heat-resistant plastic staining racks or jars
· Water bath
· Thermometer· Xylene· 95% Ethanol· 100% Ethanol· Washing container or bottle· Washing solution (PBS)

Subclass of Primary Antibody: IgG1
Immunogen of Primary Antibody: Recombinant protein consisting of part ofthe intracellular amino acid sequence (419－520) of the NPM-ALK transcript
exhibiting tyrosine kinase activity
Source of Primary Antibody: Culture supernatant of hybridoma

PBS (Phosphate-buffered saline, pH 7.6 ± 0.2)
NaCl
7.75 g1.50 gK2HPO40.20 g
KH2PO4
Distilled water 1 L-1-

· Purified water· Counterstaining solution (Hematoxylin)· Mounting media (Permanent mounting media)· Test tubes
・Slide stand· Humidified chamber for slide incubation (Moist chamber)· Cover glass· Tissue paper· Light microscope(4) Addition and Reaction of Peroxidase-Labeled Empower Reagent
1) Remove excess fluid around tissue sections on the slides carefully.
2) Apply 2 drops (100 µl) of Peroxidase-Labeled Empower Reagent,covering tissue sections completely.
3) Incubate slides in moist chamber at room temperature for 30
minutes.
4) Rinse slides in PBS (3 minutes in each washing container twice, or
use a washing bottle).

(2) Reagent Preparation
1) Preparation of ALK Antigen Retrieval Solution
Measure 1 part ALK Antigen Retrieval Solution A to 1 part ALK
Antigen Retrieval Solution B to 8 parts distilled water and mix
these well, and place gently in heat-resistant Coplin jar.
Note: Prepare required quantity accordingly and discard after use.
Do not apply used solution repeatedly.
2) Preparation of Substrate Solution
Prepare Substrate Solution just before use.
Add 1 drop of Chromogen Substrate and Substrate Buffer Solution,
respectively, to 1 ml purified water and mix well. Then, add 1 dropof Chromogen Reagent and mix again. Usage within 30 minutes ofpreparation is recommended.
3) Other reagents
Apply as supplied.

(5) Addition and Reaction of Substrate Solution
1) Remove excess fluid around tissue sections on the slides carefully.
2) Apply 2 drops (100 µl) of Substrate Solution prepared in advance,covering tissue sections completely.
3) Incubate slides in moist chamber at room temperature for 10
minutes.
4) Rinse slides with purified water.
(6) Counterstaining
1) Immerse slides in counterstaining solution.
2) Wash slides under running water.
(7) Mounting
Mount tissue sections with permanent mounting media after
dehydration and clearing in xylene.

INTERPRETATION OF STAINING RESULTS(3) Deparaffinization and Rehydration
1) Treatment with xylene(a) Immerse the slides in xylene for 3 minutes.
(b) Remove excess fluid and immerse in xylene for 3 minutes.
(c) Remove excess fluid and immerse in fresh xylene for 3 minutes.
2) Treatment with ethanol(a) Immerse in 100% ethanol for 3 minutes.
(b) Remove excess fluid and immerse in fresh 100% ethanol for 3
minutes.
(c) Remove excess fluid and immerse in 95% ethanol for 3 minutes.
(d) Remove excess fluid and immerse in fresh 95% ethanol for 3
minutes.
3) Washing
Remove excess fluid and rinse well with PBS (3 minutes in eachwashing container twice, or use a washing bottle).

1. Procedure

Examine slides under light microscope for positive staining results.
Interpret staining results by comparing with the following 3 control
slides:a. Positive Control Slide
ALK protein overexpression in tumor cells as positive result isobserved.
b. Negative Control Slide
ALK protein overexpression in tumor cells as positive result is notobserved.
c. Reagent Control Slide
ALK protein overexpression in tumor cells as positive result is notobserved. There is also considerable non-specific reaction by
non-specific protein binding when this slide is stained.
Caution(4) Treatment with high-temperature epitope unmasking
1) Preheat water bath to 95–99°C.
Note: Avoid burn injuries.
2) Prepare ALK Antigen Retrieval Solution in heat-resistant plastic
staining jar and heat to 95–99 °C in water bath. Close lid loosely toprevent excess water evaporation (Avoid sealing completely or jar
may be damaged).
3) Immerse slides in the preheated solution and close the lid loosely.
4) Incubate slides at 95–99°C for 40 minutes after verifying thattemperature of the solution is elevated to 95–99°C by thermometer.
5) Remove jar from water bath and remove the lid. Leave slides in the
jar at room temperature, 15–25 °C, for 20 minutes to cool slowly.
6) Remove slides from the solution and rinse them in PBS (3 minutesin each washing container twice, or use a washing bottle).

This kit reacts not only with ALK fusion proteins but also with thefull-length ALK protein. That is why this kit shows slight positivity or
positivity for tumors*1 that rarely express the full-length ALK protein.
This kit cannot distinguish between ALK fusion proteins and thefull-length ALK protein. Therefore, in samples, which show apositivity with this kit, a confirmation of the presence or absence of
ALK fusion genes by using the fluorescence in situ hybridization(FISH) or RT-PCR method is preferable.
*1: Large-cell neuroendocrine carcinomas of the lung, small-cell lung carcinomas, and also
rhabdomyosarcomas (particularly alveolar rhabdomyosarcomas).

2. Notes for Interpretationa. Interpret staining results consistently by comparing the staining
results of each specimen control slide.
b. Complete removal of embedding agent is important to achieve clear
staining. Residual paraffin causes strengthening of backgroundstaining.
c. False positive results may be observed in general due tonon-immunological binding of proteins and substrate reaction
products. False positive results are caused by red blood cells andmay be caused by reaction of peroxidase or reaction of endogenous
peroxidase by cytochrome C as well.
d. Interpret staining results of necrotized areas of specimen tissue
carefully by comparing with negative control slides, as it is easy forthe antigen to bind non-specifically, which is liable to be a cause ofnon-specific staining.
e. Interpret staining results carefully by comparing with negative
control slides, as the interstitial line of collagen possesses
hydrophobic properties after fixing and is easily bound with
antibody, and positively charged antibody, negatively charged for
its own, which is liable to be a cause of non-specific staining.
f. Some granulocytes and macrophages possess Fc receptors on theircytomembrane surfaces; these are likely to bind with the Fc domainof the antibody. Interpret staining results carefully by consistently
comparing with negative control slides, as some staining may befound away from the original, specific reaction site of the antibody.

5. Staining Procedure(1) Treatment with Blocking Reagent, 3 V/V% Hydrogen peroxide
solution (removal of endogenous peroxidase)
1) Remove excess fluid around tissue sections on the slides carefully.
2) Apply 2 drops (100 µl) of Blocking Reagent to each slide, coveringtissue sections completely; incubate in moist chamber at room
temperature for 5 minutes.
3) Rinse slides in PBS (3 minutes in each washing container twice, or
use a washing bottle).
(2) Addition and Reaction of Primary Antibody, Anti-human ALK
Mouse Monoclonal Antibody (5A4), or Negative Control, Mouse IgG1) Remove excess fluid around tissue sections on the slides carefully.
2) Apply 2 drops (100 µl) of Primary Antibody on specimen, Positive
Control, and Negative Control Slides, covering tissue sections
completely. For the Reagent Control Slide, apply 2 drops (100 µl) of
Negative Control instead of Primary Antibody.
3) Incubate slides in moist chamber at room temperature for 30
minutes.
4) Rinse slides in PBS (3 minutes in each washing container twice, or
use a washing bottle).
(3) Addition and Reaction of Bridge Reagent
1) Remove excess fluid around tissue sections on the slides carefully.
2) Apply 2 drops (100 µl) of Bridge Reagent, covering tissue sections
completely.
3) Incubate slides in moist chamber at room temperature for 15
minutes.
4) Rinse slides in PBS (3 minutes in each washing container twice, or
use a washing bottle).

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CAUTIONS

Problem

1. Cautions for HandlingThebackgrounds
are intensively
stained in all
the slides.

a. Handle specimens with adequate care due to the possibility of
infection risk.
b. Handle specimen tissues with adequate care due to the possibilityof infection risks such as HIV and HBV.
c. Avoid contact of reagents with skin.
d. Due to its mutagenicity, handle the Chromogen Substrate,
3,3′-Diaminobenzidine·4HCl, with adequate care .
e. Due to the hydrogen peroxide solution contained, handle theBlocking and Chromogen Reagents with adequate care.

2. Notes for Usea. Bring all reagents to room temperature (15–25°C) before use.
b. Do not use reagents after their expiry dates.
c. Do not allow tissue sections to dry out at any point during staining.
Placing tissue sections in a moist chamber during incubation withreagents is sufficient to prevent drying.
d. Replace xylene and ethanol used for deparaffinization every 40
slides.
e. Do not allow paraffin temperatures to exceed 58°C for embedding
tissues due to antigens being sensitive to heat.
f. Do not use reagents mixed with different lots, different lots,
manufacturers, or as combinations.

During thereaction, tissue
sections detachfrom the slides.

3. Waste Managementa. Primary Antibody and Negative Control should be disposed of byflushing down the drain with copious amounts of water, as there is arisk of explosion by reaction of their sodium azide content with thecopper/lead in drainpipes.
b. Prepared Substrate Solution containing DAB, as well as unused
solutions and components should be disposed of as hazardous
chemicals in accordance with all applicable rules and regulations.
c. Apparatus, reagents, and reagent containers that come in contact
with specimen tissues should be sterilized in an autoclave at 120°C
for 20 minutes or immersed in disinfectant such as 1 V/V%
hypochlorous acid overnight due to infection risks.

Problem

The unknown
specimen slide
is not stained
while thepositive control
slide is stained.

Possible cause
1. Tissue section is dry.

4. Inadequate
incubation of the
enzyme and antibody.

1) Change stale
Chromogen/Substrate Reagent.
2) Wipe off excess fluid thoroughly ateach stage.
3) Provide sufficient time forreaction with antibody. In
particular, Primary Antibody
should be incubated for the period
specified in the package insert.

1. Antigen is denatured
or masked during
fixing or embedding
process.

1) Some antigens are sensitive tofixation or embedding. Use lesspotent fixative and decrease fixingtime.

2. Antigen isdecomposed byautolysis.

1) Use tissues obtained by biopsy or
surgery whenever possible.

3. Less antigen ispresent in thesections.

1) Prolong the incubation time.

1) Obtain fresh tissues wheneveravailable.

3. Insufficient removalof paraffin.

1) Change xylene or ethanol as thecase may be.

4. Insufficient washingof antibody.

1) Ensure thorough washing ofantibody.

5. A high room
temperatureaccelerates enzyme
reactions.

1) Keep room temperature at
15–25℃.
2) Shorten reaction time.

6. Drying-out ofspecimens during
staining after
addition of thereagents.

1) Never allow the tissue to dry out.

1. Some antigens
require heat-induced
antigen retrieval
procedures or
prolonged reaction
time with thePrimary Antibody,which may cause thesections to detacheasily.

1) Mount tissue sections on slides
coated with an adhesive such as
0.02% poly-L-lysine or silane.

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 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

2. The embedding agent 1) Select a suitable embedding agent
or remove paraffin thoroughly fromis not suitable, or
embedded tissue.
paraffin has not beenthoroughly removed
2) Change xylene or ethanol solution
fromas the case may be.
paraffin-embedded
sections.
1) Adjust temperature of Antigen
Unmasking Solution to 95－99℃.
2) Incubate for 40 minutes for
accurate reaction.

2. Autolysis results inexcessive antigens
isolated inhistological solutions.

· Storage: Store at 2–8°C· Expiration: Indicated on the labels of each vial

Solution
1) Place tissue section in moist
chamber after moisturizing toprevent drying.

3. Insufficient antigen
unmaskingtreatment(high-temperature
epitope unmasking )or antibody reaction.

Solution
1) Ensure that the procedure forquenching of endogenous
peroxidase is correct.

STORAGE AND EXPIRATIONTROUBLE SHOOTING
No staining or
only weakstaining on the
Positive Control
slide and theunknown
specimen slide.

Possible cause
1. Endogenous enzyme
activity was notcompletely blocked.

INTELLECTUAL PROPERTYThis product is covered under U.S. Patent No. 6,696,548 and related
patents.

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