JP6468961B2 - 2重染色キット - Google Patents
2重染色キット Download PDFInfo
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- JP6468961B2 JP6468961B2 JP2015132308A JP2015132308A JP6468961B2 JP 6468961 B2 JP6468961 B2 JP 6468961B2 JP 2015132308 A JP2015132308 A JP 2015132308A JP 2015132308 A JP2015132308 A JP 2015132308A JP 6468961 B2 JP6468961 B2 JP 6468961B2
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- staining
- antibody
- cytokeratin
- immunostaining
- victoria blue
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- 238000012137 double-staining Methods 0.000 title claims description 11
- 238000010186 staining Methods 0.000 claims description 38
- JEVGKYBUANQAKG-UHFFFAOYSA-N victoria blue R Chemical compound [Cl-].C12=CC=CC=C2C(=[NH+]CC)C=CC1=C(C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 JEVGKYBUANQAKG-UHFFFAOYSA-N 0.000 claims description 13
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- 102100037265 Podoplanin Human genes 0.000 description 45
- 101000600766 Homo sapiens Podoplanin Proteins 0.000 description 40
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- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 26
- 208000005718 Stomach Neoplasms Diseases 0.000 description 26
- 206010017758 gastric cancer Diseases 0.000 description 26
- 230000009545 invasion Effects 0.000 description 26
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 208000008839 Kidney Neoplasms Diseases 0.000 description 10
- 206010038389 Renal cancer Diseases 0.000 description 10
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- 239000000427 antigen Substances 0.000 description 9
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- 238000000034 method Methods 0.000 description 6
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- 108010008886 CAM 5.2 antigen Proteins 0.000 description 5
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- 239000008399 tap water Substances 0.000 description 5
- 235000020679 tap water Nutrition 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
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- 208000002918 testicular germ cell tumor Diseases 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
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- 101000975474 Homo sapiens Keratin, type I cytoskeletal 10 Proteins 0.000 description 1
- 101000994455 Homo sapiens Keratin, type I cytoskeletal 23 Proteins 0.000 description 1
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
1.脱パラフィン
(i)薄切切片スライドを乾燥させる。
(ii)70℃孵卵器にてパラフィン融解(10分以上)
(iii)Tissue Tek Prisma(サクラファインテック社製)使用
-1. 乾燥::1分×1
-2. キシレン:5分×1
キシレン:3分×3
-3. 100%アルコール(ドライゾール):3分×1
100%アルコール(ドライゾール):2分×2
-4. 水洗(水道水):1分×1
-5. 終了(蒸留水中)
2.ビクトリアブルー染色(用手法)(ビクトリア青:武藤化学)
-1. 70%アルコール::3分
-2. ビクトリアブルー染色液:4時間〜over night
-3. 70%アルコール::3分まで(青色の染色性を見ながら)
-4. 水洗(水道水)
-5. 終了(蒸留水中)
3.HE染色
(GMヘマトキシリン:武藤化学、ピュアエオジン:武藤化学、エオジン希釈液:95%アルコール)
Tissue Tek Prisma(サクラファインテック社製)使用
-1. 水洗(水道水):30秒
-2. GMヘマトキシリン液:1分
-3. 水洗(水道水):30秒
-4. 温水:1分
-5. 水洗(水道水):3分
-6. 100%アルコール::15秒×2
-7. エオジン液(8倍希釈):30秒
-8. 100%アルコール::15秒×2
100%アルコール::30秒×1
100%アルコール::1分30秒×1
100%アルコール::2分×1
-9. キシレン::2分×2
キシレン::3分×1
4.IHC
(ロッシュ社製・自動免疫染色装置Ventana BenchMark XT)使用
-1. スライド(EZPrep)75℃:4分
-2. リンス(EZPrep)×2
-3. スライド(EZPrep)76℃:4分
-4. リンス(EZPrep)×1
-5. スライド常温に戻す
-6. conditioning(Cell Conditioner Medium):8分
-7. スライド(Cell Conditioner Medium)95℃:8分
-8. conditioning(Cell Conditioner Medium):30分
-9. スライド(Cell Conditioner Medium中)100℃:4分
-10. conditioning(Cell Conditioner Medium中):60分
-11 スライド常温に戻す
-12. リンス(Reaction Buffer)×2
-13. スライド(Reaction Buffer)37℃:4分
-14. リンス(Reaction Buffer)×1
-15. スライド(I-VIEW INHIBITOR)(37℃):4分
-13. リンス(Reaction Buffer)×1
-14. スライド(Reaction Buffer)37℃:4分
-15. リンス(Reaction Buffer)×1
-16. スライド(一次抗体)37℃:32分
-17. リンス(Reaction Buffer)×1
-18 スライド(Reaction Buffer)37℃:4分
-19. リンス(Reaction Buffer)×1
-20. スライド(I-VIEW BIOTIN Ig)(37℃):8分
-21. リンス(Reaction Buffer)×1
-22. スライド(I-VIEW SA-HRP)(37℃):8分
-23. リンス(Reaction Buffer)×2
-24. スライド(I-VIEW DAB and I-VIEW H2O2)(37℃):8分
-25. リンス(Reaction Buffer)×1
-26. スライド(I-VIEW COPPER)(37℃):4分
-27. リンス(Reaction Buffer)×1
-28. スライド(HEMATOXYLIN II)(37℃):16分
-29. リンス(Reaction Buffer)×2
-30. スライド(BLUING REAGENT)(37℃):4分
-31. リンス(Reaction Buffer)×2
(ビクトリアブルーHE染色併用IHCでは、上記1〜27まで)
1倍の一次抗体濃度は下記であった。
AE1/AE3抗体(DAKO社製・clone:AE1/AE3)マウスモノクローナル:610μg/L
CK18抗体(DAKO社製・clone:DC10)マウスモノクローナル:12mg/L
CAM5.2抗体(Leica社製・clone:CAM5.2)マウスモノクローナル:希釈済
図1は、胃癌症例におけるビクトリアブルーHE染色である。ビクトリアブルー染色により動静脈壁が視認可能であった。静脈内への腫瘍細胞の浸潤は認識不可能であった。実線は動脈を示し、破線は静脈を示す。
Claims (6)
- 抗サイトケラチン抗体及び抗ポドプラニン抗体を含むカクテル抗体と、弾性線維染色剤と、を有することを特徴とする2重染色キット。
- 前記弾性線維染色剤は、ビクトリアブルー染色試薬及びヘマトキシリン−エオジン染色試薬を含むことを特徴とする請求項1に記載の2重染色キット。
- 前記抗サイトケラチン抗体は、AE1/AE3及びCK18の少なくとも何れか一方を含むことを特徴とする請求項1又は2に記載の2重染色キット。
- 前記抗ポドプラニン抗体は、抗D2-40抗体であることを特徴とする請求項1乃至3の何れか1項に記載の2重染色キット。
- 前記抗サイトケラチン抗体の濃度は10〜3000μg/Lであり、前記抗ポドプラニン抗体の濃度は40〜2500μg/Lであることを特徴とする請求項1乃至4の何れか1項に記載の2重染色キット。
- 前記AE1/AE3の濃度は10〜3000μg/Lであり、前記CK18の濃度は0.1〜50mg/Lであることを特徴とする請求項3に記載の2重染色キット。
Priority Applications (1)
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JP2015132308A JP6468961B2 (ja) | 2015-07-01 | 2015-07-01 | 2重染色キット |
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JP2015132308A JP6468961B2 (ja) | 2015-07-01 | 2015-07-01 | 2重染色キット |
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Publication Number | Publication Date |
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JP2017015550A JP2017015550A (ja) | 2017-01-19 |
JP6468961B2 true JP6468961B2 (ja) | 2019-02-13 |
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JP2015132308A Active JP6468961B2 (ja) | 2015-07-01 | 2015-07-01 | 2重染色キット |
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JP (1) | JP6468961B2 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113008649A (zh) * | 2021-03-26 | 2021-06-22 | 河南赛诺特生物技术有限公司 | 一种免疫组化联合弹性纤维多重染色试剂盒、染色方法及应用 |
CN116068189A (zh) * | 2023-04-07 | 2023-05-05 | 吉林重明生物科技有限公司 | 早期癌检测试剂及其应用 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20060111898A (ko) * | 2003-08-29 | 2006-10-30 | 교와 가부시키가이샤 | 지질-풍부-플라크의 안정화 방법 및 파열 예방방법 |
GB201102189D0 (en) * | 2011-02-08 | 2011-03-23 | King S College London | Materials and methods relating to cardiovascular imaging |
CN105143847B (zh) * | 2013-02-05 | 2019-05-28 | 三路影像公司 | 细胞染色组合物及其用途 |
EP3040724B1 (en) * | 2013-09-26 | 2018-08-29 | Konica Minolta, Inc. | Method for determining quantity of biological material in tissue section |
EP3086110A4 (en) * | 2013-12-18 | 2017-06-21 | Konica Minolta, Inc. | Image processing device, pathological diagnosis support system, image processing program, and image processing method |
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