A103 : Normal tissues, more than single spot

A103 : Normal tissues, more than single spot

Specification :

•Specimen : formalin-fixed, paraffin-embedded 1.0mm diameter 54 different t

ypes of normal tissue cores. 2 spots for each tissue type. •Packing status : Each array slide is individually packed in i) a hard plastic c

ase and ii) an opaque aluminum bag sealed under a nitrogen atmosphere to

prevent oxidation and drying. •Enclosed documents: Product specification (specification, layout, coordina

tes of tissue spots : 5 pages), QC sheet (5 pages), Certification sheet (1 pag

e), General protocols (3 pages). •3.5 inch diskette (or CD) : This contains a more detailed data set related to the tissues on the slide, in MS Excel format.

Storage and handlingShipped at room temperature. Recommended storage conditions upon arrival

: 2-8 ℃. Each individually packed aluminum foil envelope has been filled with

nitrogen gas. For maximum antigenicity, use the slide as soon as possible aft

er opening.

For research use only(formalin fixed)

PETATMArrayTNV00002P .Section No.

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A B C D E F G H I

A B C D E F G H I

Layout

PE

TA

TMA

rrayA

103 .Section N

o.

1 2 3 4 5 6 7 8 9 10 11 12A

B

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I

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1 2 3 4 5 6 7 8 9 10 11 12 A1 A2 A3 A4 A5 A6

B1 B2 B3 B4 B5 B6

C1 C2 C3 C4 C5 C6

A8A7

B8B7

C8C7

A9 A10 A11 A12

D8D7

E8E7

F8F7

B9 B10 B11 B12

C9 C10 C11 C12

D9 D10 D11 D12

E9 E10 E11 E12

F9 F10 F11 F12

A

B

C

D

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D1 D2 D3 D4 D5 D6

E1 E2 E3 E4 E5 E6

F1 F2 F3 F4 F5 F6

G1 G2 G3 G4 G5 G6

H1 H2 H3 H4 H5 H6

I 1 I 2 I 3 I 4 I 5 I 6

G8G7

H8H7

G9 G10 G11 G12

H9 H10 H11 H12

A

B

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II8I7 I9 I10 I11 I12



Coordinates of tissue spots

NO. Specimen 병리번호Sex Age pathology diagonosis

1 A 1 Cerebral cortex KA020518 A1M 64 non-neoplastic2 A 2 Cerebral cortex M 64 non-neoplastic3 A 3 Cerebellum KA020518 A8M 64 non-neoplastic4 A 4 Cerebellum M 64 non-neoplastic5 A 5 Basal ganglia KA020518 A2M 64 non-neoplastic6 A 6 Basal ganglia M 64 non-neoplastic7 A 7 Hippocampus KA020518 A6M 64 non-neoplastic8 A 8 Hippocampus M 64 non-neoplastic9 A 9 Spinal cord A02-5 M 0 non-neoplastic

10 A 10 Spinal cord M 1 non-neoplastic11 A 11 Peripheral nerve 부검N1F 29 non-neoplastic12 A 12 Peripheral nerve F 30 non-neoplastic13 B 1 Spleen S02-5654 B2F 1 non-neoplastic14 B 2 Spleen F 1 non-neoplastic15 B 3 Tonsil S02-15234 BF 44 non-neoplastic16 B 4 Tonsil F 44 non-neoplastic17 B 5 Thymus S00-89 F 45 non-neoplastic18 B 6 Thymus F 45 non-neoplastic19 B 7 Fat S02-7929 MN1M 70 non-neoplastic20 B 8 Lymph node M 70 non-neoplastic21 B 9 Blood vessel(Endothelium)S02-15280 TM 22 non-neoplastic22 B 10 Blood vessel(Endothelium) M 22 non-neoplastic23 B 11 Skin S02-11511 B3F 49 non-neoplastic24 B 12 Skin F 49 non-neoplastic25 C 1 Breast S02-12604 A8F 38 non-neoplastic26 C 2 Breast F 38 non-neoplastic27 C 3 Salivary gland S02-10106 T1M 41 non-neoplastic28 C 4 Salivary gland M 41 non-neoplastic29 C 5 Thyroid gland S01-312 CF 48 non-neoplastic30 C 6 Thyroid gland F 48 non-neoplastic31 C 7 Adrenal cortex S02-4138 DF 67 non-neoplastic32 C 8 Adrenal cortex F 67 non-neoplastic33 C 9 Adrenal medulla S03-9448 ADF 63 non-neoplastic34 C 10 Adrenal medulla F 63 non-neoplastic35 C 11 Pancreas S02-5654 B1F 1 non-neoplastic36 C 12 Pancreas F 1 non-neoplastic37 D 1 Heart KA020518 A10M 64 non-neoplastic38 D 2 Heart M 64 non-neoplastic39 D 3 Lung S02-4949 A11M 69 non-neoplastic40 D 4 Lung M 69 non-neoplastic41 D 5 Bronchus S02-4949 BM 69 non-neoplastic42 D 6 Bronchus M 69 non-neoplastic43 D 7 Liver S02-14451 A5M 55 non-neoplastic44 D 8 Liver M 55 non-neoplastic

Coordinate





45 D 9 Bile duct S03-15011 A4F 46 non-neoplastic46 D 10 Bile duct F 46 non-neoplastic47 D 11 Gallbladder S02-14391 A3F 31 non-neoplastic48 D 12 Gallbladder F 31 non-neoplastic49 E 1 Kidney (Corterx) S02-16576 TF 54 non-neoplastic50 E 2 Kidney (Corterx) F 54 non-neoplastic51 E 3 Kidney (Medulla) S02-16576 TF 54 non-neoplastic52 E 4 Kidney (Medulla) F 54 non-neoplastic53 E 5 Ureter S02-10121 A1M 18 non-neoplastic54 E 6 Ureter M 18 non-neoplastic55 E 7 Bladder S02-10523 A10,19M 45 non-neoplastic56 E 8 Bladder M 45 non-neoplastic57 E 9 Prostate S02-11584 T1M 52 non-neoplastic58 E 10 Prostate M 52 non-neoplastic59 E 11 Testis S02-15191 TM 20 non-neoplastic60 E 12 Testis M 20 non-neoplastic61 F 1 Ovary S02-4150 DF 27 non-neoplastic62 F 2 Ovary F 27 non-neoplastic63 F 3 Uterus (Excervix) S02-15320 A2F 45 non-neoplastic64 F 4 Uterus (Excervix) F 45 non-neoplastic65 F 5 Uterus (Endocervix) S02-15320 A2F 45 non-neoplastic66 F 6 Uterus (Endocervix) F 45 non-neoplastic67 F 7 Uterus (Pro-Endometrium)S02-14945 B2F 44 non-neoplastic68 F 8 Uterus (Pro-Endometrium) F 44 non-neoplastic69 F 9 Uterus (Sec-Endometrium)S02-9828 B2F 53 non-neoplastic70 F 10 Uterus (Sec-Endometrium) F 53 non-neoplastic71 F 11 Uterus (Decidua) S02-14080 B3F 32 non-neoplastic72 F 12 Uterus (Decidua) F 32 non-neoplastic73 G 1 Uterus (Myometrium) S03-14702 B1F 71 non-neoplastic74 G 2 Uterus (Myometrium) F 71 non-neoplastic75 G 3 Fallopian tube F02-1656 N1F 34 non-neoplastic76 G 4 Fallopian tube F 34 non-neoplastic77 G 5 Placenta (Amnio) S02-7663 NF 40 non-neoplastic78 G 6 Placenta (Amnio) F 40 non-neoplastic79 G 7 Placenta (Chorion villi) S02-7663 NF 40 non-neoplastic80 G 8 Placenta (Chorion villi) F 40 non-neoplastic81 G 9 Placenta (Basal plate) S02-7663 NF 40 non-neoplastic82 G 10 Placenta (Basal plate) F 40 non-neoplastic83 G 11 Umbilical cord S02-14987 BF 29 non-neoplastic84 G 12 Umbilical cord F 29 non-neoplastic85 H 1 Esophagus S02-10504 BF 42 non-neoplastic86 H 2 Esophagus F 42 non-neoplastic87 H 3 Intestinal metaplasia S03-12468 ALF 37 non-neoplastic88 H 4 Intestinal metaplasia F 37 non-neoplastic

Coordinate





89 H 5 Stomach(Fundic gland) S03-7514 AFF 46 non-neoplastic90 H 6 Stomach(Fundic gland) F 46 non-neoplastic91 H 7 Stomach(Pyloric gland) S03-14983 A9M 66 non-neoplastic92 H 8 Stomach(Pyloric gland) M 66 non-neoplastic93 H 9 Duodenum S02-809 DUF 65 non-neoplastic94 H 10 Duodenum F 65 non-neoplastic95 H 11 Jeuni S02-15252 A12M 66 non-neoplastic96 H 12 Jeuni M 66 non-neoplastic97 I 1 Ileum F02-1022 CM 46 non-neoplastic98 I 2 Ileum M 46 non-neoplastic99 I 3 Appendix S98-10792 M 43 non-neoplastic

100 I 4 Appendix M 43 non-neoplastic101 I 5 Colon S02-4249 A8M 36 non-neoplastic102 I 6 Colon M 36 non-neoplastic103 I 7 Rectum S02-8112 T2M 69 non-neoplastic104 I 8 Rectum M 69 non-neoplastic105 I 9 Cecum S03-848 CM 46 non-neoplastic106 I 10 Cecum M 46 non-neoplastic107 I 11 Skeletal muscle S02-8511 T2M 59 non-neoplastic108 I 12 Skeletal muscle M 59 non-neoplastic

Coordinates



QC sheet_LOT#112120309251Haematoxylin and Eosin staining

A1 A2 A3 A4

A5 A6 A7 A8

A9 A10 A11 A12

B1 B2 B3 B4

B5 B6 B7 B8

B9 B10 B11 B12

C3 C4



C1 C2 C3 C4

C5 C6 C7 C8

C9 C10 C11 C12

D1 D2 D3 D4

D5 D6 D7 D8

D9 D10 D11 D12

E5 E6 E7 E8




E1 E2 E3 E4

E5 E6 E7 E8

E9 E10 E11 E12

F1 F2 F3 F4

F5 F6 F7 F8

F9 F10 F11 F12




G1 G2 G3 G4

G5 G6 G7 G8

G9 G10 G11 G12

H1 H2 H3 H4

H5 H6 H7 H8

H9 H10 H11 H12




I 1 I 2 I 3 I 4

I 5 I 6 I 7 I 8

I 9 I 10 I 11 I 12




Appendix: application protocol 1 Deparffinizing and H&E stain

Deparaffinization and hydration

Dry the slide at 58℃ for 1hr or overnight, before deparaffinization (put slides in horizontal position)

① Xylene (removal of paraffin) 4 X 10 min ↓②100% Ethanol (de xylene) 95% Ethanol 1min 95% Ethanol 1min 80% Ethanol 1min 70% Ethanol 1min ↓③ Wash (tap water) until washing is completed (5 min)

Routine H&E stain ① Wash (tap water) until washing is completed (5 min) ↓ ② Hematoxylin (Harris,nucleus staining,over staining) 3 min ↓ ③ Wash (tap water) ↓ ④ Decolor in 0.1% HCl, 70% Ethanol : repetitive dipping

↓ ⑤ Neutralization 10 min (tap water 5min/ Ammonia water repetitive dipping) ↓ ⑥ Eosin (cytoplasm staining) 1 min ↓ ⑦ Washing 30 sec ↓ ⑧ 70% Ethanol 1 min 80% Ethanol 1 min 95% Ethanol 1 min 95% Ethanol 1 min 100% Ethanol 1 min ↓ ⑨ Xylene (clear to increase refractive index to 1.5 fold) 4 X 10 min ↓ ⑩ Mount with mounting solution ( eg. balsam)

For research use only

IHC(immunohistochemistry)

Immunohistochemistry is an exquisitely sensitive method for locating an antigen within a cell or tissue through a high-resolution image (a single cell among thousands or millions). The method is based on the use of a primary antibody binding specifically to its cognate antigen. The bound antibody is then visualized by colorimetric or fluorescent detection methods.

Appendix: application protocol 2 Immunohistochemistry

Antigen retrieval methodDuring the preparation of tissues for staining, During the preparation of tissues for staining, antigens are heavily modified by the fixatives antigens are heavily modified by the fixatives frequently on free amino acid groups. frequently on free amino acid groups. Because they can be hidden by other Because they can be hidden by other molecules, antigen retrieval procedure is molecules, antigen retrieval procedure is required to counter these changes.required to counter these changes.There are several methods for antigen There are several methods for antigen retrieval. The selection is made according to retrieval. The selection is made according to the experimental purposes. If the experiment the experimental purposes. If the experiment is the conditioning process with first trial of is the conditioning process with first trial of that antibody, various methods need to be that antibody, various methods need to be tried. tried.

IHC procedure

The protocol needs to be optimized for The protocol needs to be optimized for antibody you may want to test and/or you antibody you may want to test and/or you might need to follow instructions from might need to follow instructions from suppliers.suppliers.

① Dry a slide at 58℃ overnight② Deparaffinize in xylene③ Hydrate the slide in gradient ethanol④ Retrieve antigen (see left section)⑤ Dip in 3% H2O2 10-15 min and washing buffer 3 X 5 min⑥ Block with normal serum

1. Proteolytic enzyme pre-treatment methodCleave the bonds formed from the fixation process.Cleave the bonds formed from the fixation process.Enzymes routinely used include trypsin, pronase or pEnzymes routinely used include trypsin, pronase or pepsin.The concentration and reaction time must be cepsin.The concentration and reaction time must be controlled since the excess enzyme treatment can daontrolled since the excess enzyme treatment can damage the target antigens. mage the target antigens.

- pronase: 0.05%(W/V) in PBS or- trypsin : 0.05%(V/V) in PBS- pepsin : 0.05%(V/V) in 2N HCl① Incubate in one of the above solutions at RT or 37℃ for 18 min② Dip in cold DW

2. Heat-induced antigen retrieval methodAntigens fixed in formalin are hidden by fixative and Antigens fixed in formalin are hidden by fixative and calcium ions. Chelating or precipitating these calciucalcium ions. Chelating or precipitating these calcium ions by specific solutions like citrate buffer, EDTA m ions by specific solutions like citrate buffer, EDTA and EGTA with heating can cleave these bonds.and EGTA with heating can cleave these bonds.① Place the slides into a rack② Immerse the slides in citrate buffer* ③ Move the entire container into microwave oven④ Microwave the slides at maximum watt for 4 X 5 min ( after each cycle, replenish any lost liquid from the slide container by addition of DW)⑤ Remove the container and allow it to cool to RT⑥ Wash with appropriate washing buffer

1. Direct method

⑦ Biotin-tagged Primary Ab for 1~2 hr at RT or 37 ℃ incubator or for overnight at 4℃(don’t wash, just change the blocking solution for primary antibody) and washing buffer 2 X 5 min

2. Indirect method

⑦-1. Primary Ab for 1~2 hr at RT or 37 ℃ incubator or for overnight at 4℃(don’t wash, just change the blocking solution for primary antibody) and washing buffer 2 X 5 min⑦-2. Biotin-tagged secondary Ab for 10-15min at RT and washing buffer 2 X 5 min

⑧ ABC reagent (streptavidin-HRP) for 10~15min and washing buffer for 2~3 X 5 min ⑨ Fresh chromogen (DAB or AEC) for 1~3 min and Wash with tap water⑩ Counter stain (the nucleus) with Hematoxylin or methyl green ⑪-1. When Hematoxylin is used : dehydrate in gradient ethanol (70% to 100%) and clear with xylene, mount with insoluble mounting medium (eg. balsam) ⑪-2. When methyl green is used : just dry and mount with soluble mounting medium (eg. glycerin or gelatin) ⑪-3. When AEC is used for chromogen : just dry and mount with soluble mounting medium

*citrate buffer : 0.01M citric acid, pH 6.0

For research use only

IHC Conditioning

Negative controls without a primary antibody, without a secondary antibody, or without detecting reagents.

Why does my negative control show strong signal?

The signal is due to non-specific cross-reactivity of detection reagents.

Possible cause of signal Trouble shooting

Primary Ab only is omitted. The secondary Ab is binding non-specifically to the tissue. Add 0.1% tissue-specific serum to the secondary Ab.

Dilute the secondary Ab.

Change species of secondary Ab.

The secondary antibody only is omitted. The detecting reagents are binding non-specifically to the tissue. Block tissue with detection reagent.

Detection reagent only is omitted. Intrinsic tissue enzyme activity is interfering with the reaction. Treat tissue with reaction solution.

Positive controls A positive antibody with the test tissue, or the test antibody with a positive tissue. Using an antibody known to react with the test tissue, or using the test antibody with cells known to contain the antigen. Fixatives may have reduced access of the antibody to the antigen. Perform microwave target retrieval procedure or protease digestion.

Why does my positive control have no signal?

In most cases, this is because the antibody is not optimized or the tissue is not adequately treated.

Possible cause of no signal Trouble shooting

Ab may be too dilute. Titrate Ab to determine the optimum dilution that gives the best signal-to-noise ratio.

Secondary Ab does not recognize the primary Ab.

Ensure secondary Ab is directed against the species of primary Ab, e.g. anti-mouse secondary Ab for mouse primary Ab

The enzyme/substrate system is defective or incompatible.

This can be confirmed by performing by dot blot test.

Documents

A103 : Normal tissues, more than single spot