A701: Test slide, various cancers plus corresponding normal Specification : (Paraformaldehyde fixed) For research use only *Specimen : Paraformaldehyde-fixed, paraffin-embedded 1.0mm diameter 12 different types of cancer tissue and matching normal tissue cores. Single spot for each tissue type. *Packing status : Each array slide is individually packed in i) a hard plastic case and ii) an opaque aluminum bag sealed under a nitrogen atmosphere to prevent oxidation and drying. *Enclosed documents: product specification (specification, layout, coordinates of tissue spots), H&E stained images, general protocols (3 pages). *3.5 inch diskette : This contains a data set related to the tissues on the slide, in MS Excel format. Storage and handling Shipped 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 after opening. Label your own PETATMArray TEST SLIDE Section No. 5 1 A B C D 2 3 4 A B C D 6 A701: Test slide, various cancers plus corresponding normal (Paraformaldehyde fixed) For research use only Layout TEST SLIDE PETATMArray Section No. A701: Test slide, various cancers plus corresponding normal (Paraformaldehyde fixed) For research use only Coordinates of tissue spot No. Coordinate Age Sex 1 29 m 1A 2B 3A 4B 5A 6B 7A 8B 9A 10 B 11 A 12 B 13 C 14 D 15 C 16 D 17 C 18 D 19 C 20 D 21 C 22 D 23 C 24 D 1 2 2 3 3 4 4 5 5 6 6 1 1 2 2 3 3 4 4 5 5 6 6 29 48 48 32 32 69 69 29 29 58 58 45 45 34 34 33 33 64 64 67 67 70 70 m f f f f m m f f f f m m f f f f m m m m m m Tissue Type Skin Skin Breast Breast Thyroid gland Thyroid gland Lung Lung Liver Liver Kidney Kidney Bladder Bladder Ovary Ovary Uterus Uterus Esophagus Esophagus Stomach Stomach Colon Colon Pathology diagnosis Squamous cell carcinoma Normal Ductal carcinoma Normal Follicular carcinoma Normal Squamous cell carcinoma Normal Hepatoma Normal Renal cell carcinoma Normal Transitional cell carcinoma Normal Serous carcinoma Fallopian tube Cervix carcinoma Normal Squamous cell carcinorma Normal Signet ring cell carcinoma Normal Adenocarcinoma Normal A701: Test slide, various cancers plus corresponding normal (Paraformaldehyde fixed) For research use only QC sheet_LOT#13101200304191 Haematoxylin and Eosin staining A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6 A701 : test slide, various cancers + corresponding normal tissues No. Age Sex Specimen Key word Pathological information A1 B1* m 29 skin Skin, scalp, excision: 1. Invasive squamous cell carcinoma, well-differentiated with extension to the invasive squamous subcutaneous fat tissue (invasion depth: about 3cm). cell carcinoma 2. Resection margins, all circumference and base: Free of tumor. 3. Lymph nodes, level 2a(0/36), level 2b(0/11), level III(0/25), level IV(0/13) and level V(0/11):(0/96): Free of tumor. Breast, right, modified radical mastectomy: 1. Infiltrating ductal carcinoma 1) Black's nuclear grade 1(poorly differentiated). infiltrating ductal 2) Modified Bloom and Richardson's histological grade III (tubule formation:3, nuclear carcinoma pleomorphism:3, mitosis:2). 3) No vascular invasion. 2. Regional lymph node, axillary(0/18), level III(0/5):(0/23): Free of tumor. Note: ER(+), PR(+), C erb B2(-). follicular carcinoma Thyroid gland, right, thyroidectomy: Follicular carcinoma, minimally invasive, showing capsular invasion. Lung, right, pneumonectomy: 1. Upper lobe: Squamous cell carcinoma, moderately differentiated, with - extension to the visceral pleura. - marked central necrosis. - tumor size: 6.3x5.5cm. - obstructive pneumonitis in the remaining lung. 2. Regional lymph nodes: Free of tumor in all nodes(0/13), in detail, subcarinal(0/6), lower paratracheal(0/2), regional(0/5). A2 B2* f 48 breast A3 B3* f 32 thyroid A4 B4* m 69 lung 1.squamous cell carcinoma 2.obstructive pneumonitis 3.pneumonia A701 : test slide, various cancers + corresponding normal tissues No. Age Sex Specimen Key word Pathological information Liver, right, lobectomy: Hepatocellular carcinoma, with 1) size: 8x7.6x7.5cm 2) Edmondson grade II 3) macrotrabecular and pseudoglandular types 4) infiltrative type 5) capsular invasion 6) necrosis: less than 5% of total volume 7) portal vein invasion 8) intact hepatic resection margin 9) Non-neoplastic liver showing congestion Kidney, left, radical nephrectomy: 1. Renal cell carcinoma, conventional(clear cell) type, with 1) Fuhrman's nuclear grade: 4 2) Foci of sarcomatoid differentiation 3) Invasion to the perinephric fat tissue but not to Gerota's fascia 4) Renal vein involvement (pT3b) 2. Lymph nodes, perihilar(0/11), paraaortic(0/2) and left common iliac(0/3): Free of tumor metastasis in all 16 nodes. Urinary bladder including prostate, seminal vesicle and ureter, radical cystectomy: Urinary bladder: Papillary urothelial carcinoma, high grade, with squamous differentiation, with various pathologic state including low grade papillary urothelial carcinoma, urothelial tumor of low malignant potential, carcinoma in situ and papilloma, with extension to the perivesical soft tissue and prostate, and with extensive lymphatic, venous and perineural invasion, incompletely excised. Ovary and fallopian tube, side unstated, salpingooophorectomy: Ovary: Borderline serous and mucinous tumor with serous micropapillary pattern (so called micropapillary serous adenocarcinoma by Kurman), confined within ovarian capsule. The matched normal tissue of this case is fallopian tube, not ovary. A5 B5* f 29 liver hepatocellular carcinoma A6 B6* f 58 kidney renal cell carcinoma C1 D1* m 45 bladder urothelial carcinoma C2 D2* f 34 ovary serous tumor borderline malignancy A701 : test slide, various cancers + corresponding normal tissues No. C3 D3* Age f Sex 33 Specimen uterus(cervix) Key word Pathological information invasive squamous Cervix: Invasive squamous cell carcinoma, large cell, keratinizing (invasion depth: 1.1cm) cell carcinoma with focal lymphovascular permeation Esophagus, esophagectomy: Basaloid squamous cell carcinoma with 1) size: 2.7x2.0x2.0cm. 2) expanding growth. 3) involvement at submucosal space and extension to upper border of proper muscle layer. 4) intact proximal and distal resection margin. 5) no tumor metastasis to upper paraesophageal lymph node (separately submitted:0/2) Stomach, subtotal gastrectomy: 1..Signet ring cell carcinoma 1. Diffuse infiltrative type 2. With extension to serosa and perigastric fat tissue(SE) 3. Frequent lymphatic permeation and perineural invasion 4. Focally mixed with tubular adenocarcinoma, moderately differentiated 5. Mixed type by Lauren's classification and infiltrative type by Ming's classification 2.. Regional lymph nodes, No.3(6/5), No.4(1/1), No.5(0/0), No.6(9/14), No.7(1/9), No.8(0/3), No.12(0/1), No.13(0/3), No.17(0/2):(17/38): Tumor metastasis in 17 out of 38 nodes. 1.Sigmoid colon, radial sigmoid colectomy: A. Adenocarcinoma, poorly differentiated, ulceroinfiltrative type with extension to pericolic fat tissue and is very close to lateral margin (about 0.5mm). B. Tubular adenoma with high grade dysplasia. Resection margins, proximal and distal: Free of tumor. 2. Regional lymph nodes, principal(0/7), pericolic(2/22):(2/29): Tumor metastasis in 2 out of 29 lymph nodes. C4 D4* m 64 esophagus basaloid carcinoma C5 D5* m 67 stomach signet ring cell carcinoma C6 D6* m 70 colon adenocarcinoma *: corresponding normal tissues Appendix: application protocol 1 Deparaffinizing and H&E stain For research use only Deparaffinization and hydration Dry the slide at 58 for 1hr or overnight, before deparaffinization (put slides in horizontal position) Xylene (removal of paraffin) 4 X 10 min 100% Ethanol (de xylene) 95% Ethanol 1min 95% Ethanol 1min 80% Ethanol 1min 70% Ethanol 1min Wash (tap water) until washing is completed (5 min) Routine H&E stain Wash (tap water) until washing is completed (5 min) Hematoxylin (Harris,nucleus staining,over staining) 3 min Wash (tap water) Decolor in 0.1% HCl, 70% Ethanol : repetitive dipping Neutralization 10 min (top water 5min/ Ammonia water repetitive dipping) Eosin (cytoplasm staining) 1 min Washing 30 sec 70% Ethanol 1 min 80% Ethanol 1 min 95% Ethanol 1 min 95% Ethanol 1 min 100% Ethanol 1 min Xylene (clear to increase refractive index to 1.5 fold) 4 X 10 min Mount with mounting solution ( eg. balsam) Appendix: application protocol 2 Immunohistochemistry 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. Antigen retrieval method During the preparation of tissues for staining, antigens are heavily modified by the fixatives frequently on free amino acid groups. Because they can be hidden by other molecules, antigen retrieval procedure is required to counter these changes. There are several methods for antigen retrieval. The selection is made according to the experimental purposes. If the experiment is the conditioning process with first trial of that antibody, various methods need to be tried. 1. Proteolytic enzyme pre-treatment method Cleave the bonds formed from the fixation process. Enzymes routinely used include trypsin, pronase or trypsin, pepsin.The concentration and reaction time must be controlled since the excess enzyme treatment can damage 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 HCl Incubate in one of the above solutions at RT or 37 for 18 min Dip in cold DW IHC procedure The protocol needs to be optimized for antibody you may want to test and/or you might need to follow instructions from suppliers. Dry a slide at 58 overnight Deparaffinize in xylene Hydrate the slide in gradient ethanol Retrieve antigen (see left section) Dip in 3% H2O2 10-15 min and washing buffer 3 X 5 min Block with normal serum 1. Direct method 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 -1. 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. Biotin-tagged secondary Ab for 10-15min at RT and washing buffer 2 X 5 min ABC reagent (streptavidin-HRP) for 10~15min and washing buffer for 2~3 X 5 min Fresh chromogen (DAB or AEC) for 1~3 min and Wash with tap water Counter stain (the nucleus) with Hematoxylin or methyl green -1. When Hematoxylin is used : dehydrate in gradient ethanol (70% to 100%) and clear with xylene, mount with insoluble mounting medium (eg. balsam) -2. When methyl green is used : just dry and mount with soluble mounting medium (eg. glycerin or gelatin) -3. When AEC is used for chromogen : just dry and mount with soluble mounting medium 2. Heat-induced antigen retrieval method Antigens fixed in formalin are hidden by fixative and calcium ions. Chelating or precipitating these calcium ions by specific solutions like citrate buffer, EDTA and EGTA with heating can cleave these bonds. Place the slides into a rack Immerse the slides in citrate buffer* Move the entire container into microwave oven 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) Remove the container and allow it to cool to RT Wash with appropriate washing buffer *citrate buffer : 0.01M citric acid, pH 6.0 IHC Conditioning Negative controls without a primary antibody, without a secondary antibody, or without detecting reagents. Why does my negative control show strong signal? Possible cause of signal Primary Ab only is omitted. The signal is due to non-specific cross-reactivity of detection reagents. Trouble shooting 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? Possible cause of no signal Ab may be too dilute. In most cases, this is because the antibody is not optimized or the tissue is not adequately treated. Trouble shooting Titrate Ab to determine the optimum dilution that gives the best signal-to-noise ratio. Ensure secondary Ab is directed against the species of primary Ab, e.g. anti-mouse secondary Ab for mouse primary Ab This can be confirmed by performing by dot blot test. Secondary Ab does not recognize the primary Ab. The enzyme/substrate system is defective or incompatible.