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Molecular Pathology Core Laboratory

Molecular Pathology Core Laboratory. PI: 戚謹文/李芬瑤 Chin-Wen Chi, Ph.D., Anna F-Y. Li, M.D.* Department of Medical Research & Education Department of Pathology* Taipei Veterans General Hospital National Yang Ming University. Specific Aims.

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Molecular Pathology Core Laboratory

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  1. Molecular Pathology Core Laboratory PI: 戚謹文/李芬瑤 Chin-Wen Chi, Ph.D., Anna F-Y. Li, M.D.* Department of Medical Research & Education Department of Pathology* Taipei Veterans General Hospital National Yang Ming University

  2. Specific Aims 1.To establish a core facility for the preparation and use of pathological samples. 2.To provide pathological services for basic researchers.

  3. Service provided (1)Dr. 張國威: preparation of wax array blocks and 8 sections per block (8 samples), preparation of array block (one block with 24 samples) (2)Dr. 范明基: immunostaining of 8 human hepatoma tissue array with 3 monoclonal antibodies. (3)Dr. 鐘明怡: provided both normal ( 2 slides) and tumor (10 slides) tissue array slides (33-38 tissues). provided both normal ( 10 slides) and tumor (10 slides) tissue array slides (9 tissues). (4)Dr. 胡承波: Wax block preparation (38 blocks), section and HE stain (2 blocks), with image files. (5)Dr. 周成功: Immunostaining of human hepatoma array slides (10 slides), with two new antibodies and their peptide blocker.

  4. The PixCell® II performs Laser Capture Microdissection (LCM) from heterogeneous tissue samples simply, quickly and precisely. In minutes, you can locate a single cell or large groups of cells and, using a simple aim-and-shoot method, extract them for subsequent molecular analysis. LCM preserves the exact morphologies of both the captured cells as well as the surrounding tissue. The PixCell II transfers cells from paraffin-embedded and frozen tissue samples, stained and immunolabelled slides. Monitor and document the entire process, and store images in the archiving workstation. Microdissect fluorescently-stained cells with the optional fluorescence package.

  5. Applications utilizing LCM Research Applications      Genomics           Differential Gene Profiling           Loss of Heterozygosity           Microsatellite Instability           Gene Quantification      Proteomics           Two-Dimensional Protein Gels           Western Blotting           Immuno-quantification of Proteins

  6. Molecular Pathology WorkshopPreparation and Staining of Tissue 戚謹文/丁令旦* Department of Medical Research & Education Department of Pathology* Taipei Veterans General Hospital National Yang Ming University

  7. 組織固定包埋及染色原理介紹 Materials used in your project: Cells. Tissues Target of your study: DNA, RNA, protein .

  8. 一、 為確保研究用檢體之正當採集及使用，保障授權人之權益，特訂定本注意事項。採集檢體供研究使用，除依法令規定外，依本注意事項為之。一、 為確保研究用檢體之正當採集及使用，保障授權人之權益，特訂定本注意事項。採集檢體供研究使用，除依法令規定外，依本注意事項為之。 • 將供研究用或非供研究用所採集之檢體，使用於教學時，不視為供研究使用。 • 二、 本注意事項用詞定義如下: • (一) 檢體 : 指採集受檢人之細胞、組織、器官、體液或其衍生物質，包括採集自與母體分離之胎兒者，但不包括採集自死後之人體者。 • (二) 受檢人﹕指接受檢體採集之人。 • (三) 檢體使用者﹕指直接使用檢體、指示他人使用檢體或依受檢人間之契約等特定關係而得使用檢體之人。 • 三、 檢體之採集與使用不得違背醫學倫理，並應注意防制對人類及生態環境之危害。 研究用人體檢體採集與使用注意事項(1) 91.01.02 DOH

  9. 採集檢體使用，除法律有規定者外，應告知受檢人下列事項，並取得其同意。受檢人未滿十七歲或無識別能力者，由其法定代理人、配偶或家屬代為同意。採集檢體使用，除法律有規定者外，應告知受檢人下列事項，並取得其同意。受檢人未滿十七歲或無識別能力者，由其法定代理人、配偶或家屬代為同意。 • (一) 採集之目的及其可能使用範圍與使用期間。 • (二) 採集之方法及數量。 • (三) 可能發生之併發症及危險。 • (四) 受檢人之權益與檢體使用者之義務。 • (五) 檢體是否有提供或轉讓他人或國外使用等情形。 • (六) 研究經費來源及所有參與研究之機構。 • (七) 其他與檢體採集或使用有關之重要事項。 • 前項告知與同意應以書面為之，必要時輔以口頭告知，使受檢人明瞭其內容。但受檢人同意不使用書面者，得不使用書面。 研究用人體檢體採集與使用注意事項(2)

  10. 一、 因採集檢體使用可能衍生其他權益時，檢體使用者應告知受檢人並為必要之書面約定。一、 因採集檢體使用可能衍生其他權益時，檢體使用者應告知受檢人並為必要之書面約定。 • 二、 檢體使用者應在受檢人所同意或依法得使用之範圍內使用檢體。使用檢體如逾越前項範圍，應依第四點規定再次告知受檢人並取得同意。 • 三、 除法律有規定者外，受檢人得拒絕接受採檢、終止檢體使用之同意或變更所同意之檢體使用範圍。受檢人拒絕接受其個人醫療使用以外之採檢者，應不影響其醫療上之權益。 • 檢體使用者應妥善保存、處置並使用檢體。使用完畢並應確實銷毀。 研究用人體檢體採集與使用注意事項(3)

  11. 一、 檢體使用者應尊重並保護受檢人之人格權。一、 檢體使用者應尊重並保護受檢人之人格權。 • 對於因檢體採集、保存、使用所知悉之受檢人秘密、隱私或個人資 料，不得無故洩漏。 • 二、 本注意事項頒行前已採集之檢體，有下列情形之一者，得不受第四點、第五點、第六點規定之限制 • :(一) 難以辨認受檢人身分。 • (二) 難以重新取得受檢人同意。 • (三)已可公開取得之檢體。 研究用人體檢體採集與使用注意事項(4)

  12. 組織固定包埋及染色原理介紹 Materials used in your project: Non-human Cells or tissues: rats, mice, rabbits, dogs, pigs, etc. Please get approval from your Institute’s 實驗動物管理委員會 (農委會40248號令; July 13, 2001) .

  13. 組織固定-FIXATION (1) What will happen to a tissue after removal from living condition? Bacteria growth Autolysis How to maintain cell or tissues structure, solid and liquid phases. .

  14. FIXATION (Time and Temperature) Tissue should be fixed ASAP after removal. If fixation can not be done right away, tissue should be kept in refrigerator until the fixative can be applied. RNAlater: do not put into refrigerator, it works at room temperature.. .

  15. Fixing solutions 1. Fast penetration 2. Coagulate cell contents into insoluble substances 3. Protect tissue from shrinkage and distortion during dehydration, embedding and sectioning. 4. Allow cellular targets for further assay such as staining or in situ hybridization. .

  16. Fixing solutions 1. Acetic acid 2. Acetone 3. Ethanol. 4. Formaldehyde 5. Osmium tetroxide 6. Picric acid. .

  17. FIXATION:special consideration Lipiodol uptake in hepatoma: Aqueous or alcoholic fixative? In situ hybridization: Formalin, paraformaldehyde, acetone. In situ perfusion fixation. Time of fixation, size of tissue. .

  18. 石蠟包埋標本 (1) 組織固定 : 組織取得後 ,先將組織切成不超過2 cm x 2 cm x 0.5 cm大小的組織塊(標本較小亦可),再放入約組織15-20倍體積的4 % Paraformaldehyde中固定 , 固定時間為 12-24小時另外可以10% Formalin固定標本. Cells, Soft vs. hard tissues.

  19. 石蠟包埋標本 (2) 組織脫水 , 清洗 , 浸潤 : 每天下午5時 , 將組織分別放入專用包埋盒內 , 置密閉式組織脫水機中進行一系列處理 , 再於隔日早上8時取出進行組織包埋 . 下列為密閉式組織脫水機之處理程序 : (密閉式組織脫水機:廠牌 SHANDON 型號 Hypercenter XP) 1. 70% alcohol 4 hrs 2. 80% alcohol 1 hr 3. 95% alcohol 1 hr 4. 95% alcohol 1 hr 5. 100% alcohol 1 hr 6. 100% alcohol 1 hr 7. 100% alcohol 1 hr 8. xylene substitute 1 hr 9. xylene substitute 1 hr 10. paraffin 1 hr 11. paraffin 2hr 組織包埋 : 每日早上8時進行組織包埋 , 組織包埋時使用經加熱溶為液態的石蠟 ,經快速冷卻使之凝固成組織蠟塊. 蠟塊切片5 mm 後以H.E. stain染色, 經病理醫師判讀後影像存檔

  20. 1. 70% alcohol 1 hrs 2. 80% alcohol 1 hr 3. 95% alcohol 1 hr 4. 95% alcohol 1 hr 5. 100% alcohol 1 hr 6. 100% alcohol 1 hr 7. 100% alcohol 1 hr 8. xylene substitute 1 hr 9. xylene substitute 1 hr 10. paraffin 1.5 hr 11. paraffin 1.5 hr 組織包埋 : 每日早上8時進行組織包埋 , 組織包埋時使用經加熱溶為液態的石蠟 ,經快速冷卻使之凝固成組織蠟塊. 蠟塊切片5 mm 後以H.E. stain染色, 經病理醫師判讀後影像存檔

  21. 石蠟包埋標本 (3) 組織包埋 : 每日早上8時進行組織包埋 , 組織包埋時使用經加熱溶為液態的石蠟 ,經快速冷卻使之凝固成組織蠟塊. 蠟塊切片5 mm 後以Hematoxylin and eosin stain染色

  22. Tissue Array Paraffin Block

  23. O.C.T.標本( TISSUE-TEK MILES Inc. 4583) 長x寬x高 = 0.7 x 0.7 x 0.7 (cm3)之標本. 大標本瓶內有DMEM粉紅色液體者(GIBCO/BRL)，貼上病人標籤，並分別在蓋上註明N或T，放入標本後請放回標本室門外冰箱中. O.C.T.標本以liquid nitrogen frozen後長期儲存於-80℃冰櫃中

  24. Sectioning Cut a thin slice of tissue (5-8 um) 1. Frozen section: 2. Wax embedded tissue: Slides: coated slides(Muto glass silane coated slides) non-coated slides (LCM) [Superfrost]

  25. Sectioning Sections of paraformaldehyde fixed, OCT-embedded tissue are sectioned at 7 to 10 mm and can be stored desiccated at -70°C until needed.

  26. Cell plating on slides To sterilize glass coverslips, dip in ethanol and flame. One can use 22x22x1 mm3 coverslips and put them in 6-well plates. Seed 100,000 cells per well overnight and fix the next day.

  27. Cell plating on slides Remove the media and rinse once with PBS. Remove the PBS and immediately add -20°C methanol. (Do not allow the cells to dry.) Put the plate in a -20°C freezer for 5 min. Remove the methanol and add PHEM buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2, pH = 6.9) Fixed cells are kept at 4°C in PHEM prior to immunostaining.

  28. Sectioning: Handling RNA Collection of Tissue for RNA Analysis : Ribonucleases (RNases) are very stable and active enzymes that degrade RNA. Minute amounts are sufficient to destroy RNA. It is important to use RNase free conditions during all apsects of tissue collection. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed. The use of sterile, disposable polypropylene tubes is recommended.

  29. Sectioning:Handling RNA Necrospy Instruments: Metal instruments (scissors, pick-ups, etc) can be autoclaved prior to use or washed with DEPC water and baked at * 240°C for 4 or more hours.Autoclave only is not enough. Glassware can be treated with 0.1% DEPC * (diethyl pyrocarbonate). Fill glassware with DEPC, allow to stand overnight (12 h) at 37°C, and then autoclave or heat to 100°C for 15 min to remove residual DEPC. Alternatively, all plastic and glass ware, instruments and work surfaces may be treated with RNase Zapo (Ambion), a RNase decontamination solution. Sample Collection of Rodent or Non-human samples: At necropsy, tissues should be collected within 12-15 minutes following death. No specific form of euthanasia is required. If the tissue collection time is much greater than 15 minutes, RNases will have been activated and it is highly unlikely that good quality RNA will be obtained for further analysis.

  30. 1. xylene substitute 10 min 2. xylene substitute 10 min 3. 100% alcohol 1 min 4. 100% alcohol 1 min 5. 95% alcohol 1 min 6. 80% alcohol 1 min 7. 70% alcohol 1 min 8. Running water 3 min 9. Harries Hematoxylin 15 min 10. Running water 2 min 11. paraffin 1.5 hr 組織包埋 : 每日早上8時進行組織包埋 , 組織包埋時使用經加熱溶為液態的石蠟 ,經快速冷卻使之凝固成組織蠟塊. 蠟塊切片5 mm 後以H.E. stain染色, 經病理醫師判讀後影像存檔

  31. 11. 0.5% acid alcohol 3 sec 12. Running water 1 min 13. Ammonia water 30 sec 14. Running water 20 min 15. 0.5% alcoholic eosin 2-5 min 16. 95% alcohol 10 sec 17. 95% alcohol 10 sec 18. 100% alcohol 30 sec 19. 100% alcohol 1 min 20. Xylene substitute 5 min 21. Seal the slides with sealer 11. paraffin 1.5 hr 組織包埋 : 每日早上8時進行組織包埋 , 組織包埋時使用經加熱溶為液態的石蠟 ,經快速冷卻使之凝固成組織蠟塊. 蠟塊切片5 mm 後以H.E. stain染色, 經病理醫師判讀後影像存檔

  32. Mounting media Organic mountant: Rapid mounting media for microscopy (Merck), or Permount Aqueous mountant: Kaiser’s glycerol gelatin (Merck). (Intracellular components such as lipid will be dissolved by organic mountant) Watch out for air bubbles.

  33. Useful website and book http://dir.niehs.nih.gov/dirlep/ http://dir.niehs.nih.gov/dirlep/immuno.html P53, COX-2, Insulin, PCNA, BrDU, ER, GST-pi, …..etc (images and protocols) Laser capture microdissection: protocols. Animal Tissue Techniques, GL Humason W. H. Freeman and Company. (美亞書店)

  34. Reagents Paraformaldehyde: Nacalai Hematoxylin, 1% Eosin Y solution : MUTO Pure Chemicals Co. Xylene substitute: Ultra Clear from J. T. Baker. Kaiser’s glycerol gelatin, Rapid Mounting media for microscopy from Merck. :

  35. Acknowledgement 1. National Yang Ming University Genome Center. 2.Departments of Surgery(General Surgery, Experimental Surgery), Pathology, and Research & Education, Taipei Veterans General Hospital. 3. National Health Research Institute

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