For research use only

IHC staining system
For detection oflower expression of ALK fusion proteinsprotALK

Anaplastic Lymphoma Kinase-Histofine® ALK Detection KIT
Code: 417071F Size: 20 tests-Histofine® ALK Detection Kit detects anaplastic lymphoma kinase (ALK) proteins intumor cells in paraffin-embedded tissue specimens by IHC staining and determines
presence of such protein expression.

Background
The ALK gene was identified in 1994 as a gene fused to the nucleophosmin (NPM) gene in anaplastic large-celllymphoma (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 intracellular domain, and its function is associated with the promotion of cell growth and inhibition ofapoptosis.
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 and SEC31L1 genes in inflammatory myofibroblastic tumor (IMT).
The proteins produced from these fusion genes are constantly activated by forming dimers and led to cancerouschange.
Recently, other ALK fusion genes with EML4 gene, KIF5B gene or KCL1 gene in non-small cell carcinoma oflung, SEC31A gene or SQSTM1 gene in ALK-positive large B-cell lymphoma and VCL gene in renal cell cancer
have been also reported.

Staining Image■ Strong Positive Image■ Weak Positive Image

Lung adenocarcinoma: Positive for cytoplasm of tumor cells. Weak to strong positive of staining
levels regarding the expression level of ALK fusion protein. Hot bath treatment (+)

Kit Components
Product Name-Histofine®

ALK Detection KitComponents
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-Histofine®

Size

Code20 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

Volume4ml × 12ml × 12ml × 14ml × 14ml × 10.5ml × 10.5ml × 1
0.5ml × 1150ml × 1150ml × 1

Product Name

Size

Code

ALK Control Slides

5 slides417081FNotes 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 areobserved. 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 (particularlyalveolar 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 (+)

References(1) Shiota M., et al: Hyperphosphorylation of a novel 80 kDa protein-tyrosine kinase similar to Ltk in a human Ki-1 lymphoma cellline, 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 ofanti-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

Manufacturer
6-19-20, TSUKIJI, CHUO-KU, TOKYO 104-8402, JAPAN
PHONE : 81-3-3248-2207
FAX: 81-3-3248-2243
http://www.nichirei.co.jp/bio/english
2012.05

-Histofine® ALK Detection KIT● Following procedure is required for accurate staining results.
● Designated temperature and duration of operation is required at each step.
● Operation under RT (15 to 25 °C) is required except designated temperature.

Staining Procedure● Preparation of tissue sections

Slice sections into 4 μm thickness and place on
poly-L-lysine or silane-coated slides.

Dry the slides completely in incubator.
(at 37 °C for 24hrs).

●Preparation of ALK Antigen Retrieval Solution
Avoid burn injuries.
ALK Antigen
Retrieval Solution A(Vial 9)thermometer

ALK Antigen
Retrieval Solution B(Vial 10)Distilled water

Measure 1 part ALK Antigen Retrieval Solution
A to 1 part ALK Antigen Retrieval Solution B to8 parts distilled water.

Preheat water bath up to 95-99 °C.
Prepare ALK Antigen Retrieval Solution
in heat-resistant plastic staining jar andheat up to 95-99 °C in the water bath.

Note:
Lids on staining jar and water bath are
effective to maintain 95 to 99 °C andto prevent excess water evaporation.
Those lids should be placed loosely to
avoid completely sealed condition orthe jar may have risk of damage.

●Deparaffinization and Rehydration (for paraffin-embedded tissue sections)xylene3 min.

xylene3 min.

xylene3 min.

100% ethanol
3 min.

100% ethanol
3 min.

95% ethanol
3 min.

95% ethanol
3 min.

PBS
3 min. x 3● Remove excess xylene and ethanol at each step.
● Change xylene and ethanol as appropriate to complete deparaffinization and rehydration.

●Antigen Retrieval
Designated temperature and duration of operation is required for accurate staining results.

Immerse the slides in 95-99 °C of ALK
Antigen Retrieval Solution and place lidon the staining jar loosely.
And place another lid on the water bathloosely then heat the solution up to
95-99 °C again.

Incubate the slides after confirming
temperature of the Solution risen to
95-99 °C by thermometers.
(at 95-99 °C for 40 min.)Note:
Lids on staining jar and water bath are
effective to maintain 95 to 99 °C and toprevent excess water evaporation.
Those lids should be placed loosely to
avoid completely sealed condition or thejar may have risk of damage.

Remove the staining jar from the water
bath and uncover the jar. Cool the jar
down slowly leaving the slides inside.
(at RT for 20 min.)●Treatment with Blocking Reagent* (Removal of endogenous peroxidase)
Absorbent
paper

Remove PBS around tissue sections.

Avoid burn injuries.

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

* 3V/V% Hydrogen peroxide solution

Blocking Reagent(Vial 1)

Apply Blocking Reagent to each slide covering
sections completely and incubate in moist chamber.
(at RT for 5 min.)

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

●Addition and reaction of Primary Antibody* or Negative Control*** anti-ALK mouse monoclonal antibody (5A4)** Mouse IgG

Primary Antibody (Vial 2)
orNegative Control (Vial 3)

Absorbent
paper

Remove PBS around tissue sections.

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

Apply Primary Antibody or Negative Control to eachslide covering sections completely and incubate inmoist chamber. (at RT for 30 min.)●Addition and reaction of Bridge Reagent
Bridge Reagent(Vial 4)

Absorbent
paper

Remove PBS around tissue sections.

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

Apply Bridge Reagent to each slide covering sectionscompletely and incubate in moist chamber. (at RT for15 min.)●Addition and reaction of Peroxidase labeled Empower Reagent
Peroxidase labeledEmpower Reagent(Vial 5)

Absorbent
paper

Remove PBS around tissue sections.

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

Apply Peroxidase labeled Empower Reagent to eachslide covering sections completely and incubate inmoist chamber. (at RT for 30 min.)●Preparation, addition and reaction of Substrate Solution
Preparation of
Substrate Solution

Substrate BufferSolution (Vial 7)

Chromogen
Substrate(Vial 6)

SubstrateSolution

Absorbent
paperChromogenReagent(Vial 8)Add each 1 drop of Chromogen Substrate andSubstrate Buffer Solution to 1mL of purified water andmix well. And then add Chromogen Reagent to the
mixture and mix again. (Use the solution within 30 min.
after preparation is recommended.)●Counterstaining●MountingRemove PBS aroundtissue sections.

Apply prepared Substrate Solution
to each slide covering sections
completely and incubate in moist
chamber. (at RT for 10 min.)Wash the slides with
distilled water.

Wash the slides with running water after immersed in counterstaining reagent (hematoxylin).

Mount slides with permanent mounting media after dehydration in graded series of alcohol and clearing in xylene.

-Histofine® ALK Control Slides
Code: 417081F Size: 5 slides-Histofine® ALK Control Slides are used as a standard for discrimination of positive tissue blocks possess
expressed ALK protein.
■ Negative control cell line/SK-BR-3 ■ Positive control cell line/NCI-H2228
This product is used for validation ofreagent performance and staining
technique in the IHC staining(including cytology staining) with-Histofine® ALK Detection Kit.
Both formalin fixed paraffin embeddedcell lines of NCI-H2228 (Positive) andSK-BR-3 (Negative) are mounted oneach slide.

No stain is observed in the cytoplasm of all cells.
Treatment with hot bath (+)

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