人乳头状瘤病毒 ( 广谱 ) 检测试剂盒 ( 原位杂交 ) 实验前需配试剂 A 1X 杂交增强液取适量 100X 杂交增强液及蒸馏水, 按 1:99 稀释, 并将此 1X 杂交增强液存放于室温备用 B 30% 湿盒增效液取湿盒增效液及蒸馏水, 按 3:7 配制成 30% 湿盒增效液备用实验时现配试

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1 人乳头状瘤病毒 ( 广谱 ) 检测试剂盒 ( 原位杂交 ) 规格 : 500ul 1250ul 编号 : S30292 S30293 ( 操作之前请详细阅读此说明书 ) 检测原理和意义原位杂交技术的原理是采用荧光生物素地高辛等标记的探针, 依据碱基互补配对原理, 与组织切片细胞涂片等标本中的靶序列特异杂交, 经信号放大及显色后, 镜下观察结果此方法具有很高的灵敏性和特异性人乳头状瘤病毒 (Human Papillomavirus,HPV) 属于乳多孔病毒科的乳头瘤病毒属其主要通过直接或间接接触污染物品或性传播感染人类, 引起人类皮肤和粘膜的多种良性乳头状瘤或疣, 某些型别的感染具致癌性目前已检出 100 多种人乳头状瘤病毒, 其中 50 种左右可以感染粘膜口腔阴道子宫颈肛周及直肠低危型如 HPV6,11 等, 常引起外生殖器湿疣等良性病变包括宫颈上皮内低度病变 (CINⅠ) 高危型如 HPV16,18,31,33 等与宫颈癌及宫颈上皮内高度病变 (CINⅡ/Ⅲ) 的发生相关, 尤以 16 和 18 型为主福州泰普生物研发的 HPV 广谱原位杂交试剂盒, 根据 HPV 广谱序列制备的探针, 涵盖了 28 种 HPV 病毒亚型, 包括 :5, 6, 8, 11, 16, 18, 26, 27, 30, 31, 33, 35, 39, 40, 41, 42, 43, 45, 47, 48, 51, 52, 53, 54, 55, 57, 58, 59, 是病理组织切片细胞培养涂片及细胞涂片原位检测 HPV 病毒的有力手段试剂盒提供的材料和试剂 ( 注 :HPV 广谱杂交液另小袋包装,-20 保存 ) 成份保存方法 500ul 提供量 1250ul 1 HPV 广谱杂交液 ul 625ulx2 2 杂交增强液 (100 ) 4 60ml 60ml 3 一抗 4 1ml 2.5ml 4 二抗 4 1ml 2.5ml 5 HRP 聚合物 4 1ml 2.5ml 6 DAB 底物 4 1ml 2.5ml 7 DAB 4 20ul 50ul 赠送 18X18mm 盖玻片 4 / 室温 20 片 50 片湿盒增效液 4 / 室温 30ml 30ml 用户自备材料和试剂 1. 原位杂交加热器 ( 泰普编号 :M10061) 8. 湿盒 2 个 2. 微波炉 9. 二甲苯恒温培养箱 10. 酒精 4. 光学显微镜 11. PBS(PH7.6) 5. 移液器 12. 苏木素 6. 计时器 13. 氨水 7. 玻片染色缸和染色架 14. 盐酸酒精公司名称 : 福建泰普生物科学有限公司总部 : 福建省福州市金山金洲北路 7 号科技企业 7 号楼 4 层邮编 : 电话 : , 传真 : 网址 : 邮箱 :tib@tibchina.com 免费电话 :

2 人乳头状瘤病毒 ( 广谱 ) 检测试剂盒 ( 原位杂交 ) 实验前需配试剂 A 1X 杂交增强液取适量 100X 杂交增强液及蒸馏水, 按 1:99 稀释, 并将此 1X 杂交增强液存放于室温备用 B 30% 湿盒增效液取湿盒增效液及蒸馏水, 按 3:7 配制成 30% 湿盒增效液备用实验时现配试剂 DAB 显色液依据需要配制 DAB 显色液, 以可覆盖整个组织的量为宜取 DAB 与 DAB 底物, 按照 1:50 的比例混匀, 避光保存备用 ( 建议每次实验同时附加阳性阴性对照片 ) 操作步骤 1. 切片处理 : 将 4um 组织切片粘附在 APES 处理的载玻片上, 切片于烘烤 60 分钟以上 2. 脱蜡 : 溶液次数时间 ( 分 ) 二甲苯 % 酒精 % 酒精 1 x 3 80% 酒精 1 3 蒸馏水 1 x 3 ( 注意 : 使用过的二甲苯, 应相应延长脱蜡时间, 以保证脱蜡完全 ) 3. 微波处理 : 将切片放入盛有 1X 杂交增强液的容器里, 微波高火状态煮 30 分钟, 自然冷却 2-3 分钟 ( 加热过程中, 溶液应始终浸没组织建议适时 ( 每隔 5-15 分钟 ) 补充杂交增强液 ) 蒸馏水洗涤 1 1 分钟, 小心擦干组织周围液体 4. 杂交 : 4.1 每张切片滴加适量杂交液 ( 约 25 ul, 根据组织大小增减量 ), 并加盖玻片 ( 本试剂盒配备, 可根据加入杂交液量进行剪裁 ); 4.2 放置于 95 原位杂交加热器中变性 5 分钟 ; 4.3 放入含 30% 湿盒增效液的湿盒中,37 下孵育 4-16 小时建议过夜 (<16 小时 ) 以保证低拷贝样本的杂交效果 ; (PBS 预温 ) 浸泡切片, 小心移去盖玻片,PBS 继续浸泡 5 分钟 ; PBS 洗涤 3x 5 分钟 ( 轻微振荡容器 ); ( 注意 : 杂交液量应与盖玻片规格匹配, 切片在整个试验过程中要保持湿润状态, 防止干片 ) 5. 信号放大与显色 : 5.1 小心擦去组织周围液体, 滴加适量一抗 ( 约 50 ul, 根据组织大小增减量 ),37, 水湿盒中孵育 30 分钟 ;37 PBS 浸泡 3x2 分钟 ( 轻微振荡容器 ); 5.2 小心擦去组织周围液体, 滴加适量二抗 ( 约 50 ul, 根据组织大小增减量 ), 室温, 水湿盒中孵育 20 分钟 ;PBS 室温浸泡 3x2 分钟 ( 轻微振荡容器 ); 5.3 小心擦去组织周围液体, 滴加适量 HRP 聚合物 ( 约 50 ul, 根据组织大小增减量 ), 室温, 水湿盒中孵育 30 分钟 ;PBS 室温浸泡 3x2 分钟 ( 轻微振荡容器 ) 公司名称 : 福建泰普生物科学有限公司总部 : 福建省福州市金山金洲北路 7 号科技企业 7 号楼 4 层邮编 : 电话 : , 传真 : 网址 : 邮箱 :tib@tibchina.com 免费电话 :

3 人乳头状瘤病毒 ( 广谱 ) 检测试剂盒 ( 原位杂交 ) 5.4 小心擦去组织周围液体, 滴加适量 DAB 显色液 ( 约 50 ul, 根据组织大小增减量 ), 室温, 水湿盒中孵育约 10 分钟 ( 不同样本的显色时间可能有所差别, 建议镜下观察显色过程 );PBS 室温浸泡 3x2 分钟 ( 轻微振荡容器 ) 5.5 自来水冲洗, 苏木精复染 ( 可用 HCl 分化及氨水返蓝 ) 梯度酒精脱水, 中性树胶封片阳性结果判断标准 : 上皮细胞核棕褐色着色为主参考文献 : 1. Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia, Journal of General Virology (2000), 81, Role of HPV DNA testing in predicting cervical intraepithelial lesions: comparison of HC HPV and ISH HPV, Diagn. Cytopathol. 2003; 29: HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears, J Histochem Cytochem 50: , Electronmicroscopic demonstration of HPV in Oral Warts, Micromologica, 13, 27-34, 1990 其他事项 : 本试剂保质期为 12 个月客户在收到产品时请及时检查, 如发现有外包装破损试剂盒成分不全等产品问题, 或者在使用过程中有任何疑问, 请及时与泰普客户服务中心联系, 我们将及时为您解决公司名称 : 福建泰普生物科学有限公司总部 : 福建省福州市金山金洲北路 7 号科技企业 7 号楼 4 层邮编 : 电话 : , 传真 : 网址 : 邮箱 :tib@tibchina.com 免费电话 :

4 人乳头状瘤病毒 ( 广谱 ) 检测试剂盒 ( 原位杂交 ) 附 : 简要操作步骤操作参数 1 二甲苯 3X % 酒精 1X3 3 95% 酒精 1X3 4 80% 酒精 1X3 5 蒸馏水 1X3 6 微波加热 3x10 7 蒸馏水 1X1 8 加杂交液适量 9 变性 95, % 湿盒增效液的湿盒中孵育 37,4-16h 11 PBS( 用前 48 预温 ) 脱片 + 浸泡 PBS( 用前 48 预温 ) 浸泡 3X5 13 加一抗 37,30 14 PBS 浸泡 37,3X2 15 加二抗室温,20 16 PBS 浸泡室温 3X2 17 加 HRP 聚合物室温,30 18 PBS 浸泡室温 3X2 19 DAB 显色室温, PBS 浸泡室温 3X2 21 自来水冲洗室温 1X1 22 复染, 封片常规公司名称 : 福建泰普生物科学有限公司总部 : 福建省福州市金山金洲北路 7 号科技企业 7 号楼 4 层邮编 : 电话 : , 传真 : 网址 : 邮箱 :tib@tibchina.com 免费电话 :

5 In Situ Hybridization Wide Spectrum HPV Test Kit Cat. No. S30292 S30293 Spec 500ul 1250ul Principle and Significance The principle of In Situ Hybridization is based on the specific hybridization between the labelled probe (biotin or digoxigenin) and target sequence (DNA or RNA sequence) in tissue and cytological specimens. The hybridism can be visualized easily by a specific staining procedure in which a combination of BCIP/NBT or DAB and HRP conjugated DIG/BIO antibodies will yield a detectable and dyed precipitation. The technique is highly sensitive and specific. Human Papillomavirus (HPV) is the name of a group of viruses. HPV can be transmitted environmentally or by casual skin-to skin contact. Some types of these viruses infect the mucous membranes and skin and can cause warts and papillomas, for which the virus family is named. Over 100 different human papillomavirus (HPV) types have been characterized. Low risk strains of human papillomavirus (HPV), such as HPV 6 and 11, cause most cases of condyloma and cervical intraepithelial neoplasia I (CIN I); High risk strains of human papillomavirus (HPV), such as HPV 16, 18, 31 and 33,have close relation to high grade cervical intraepithelial neoplasia II/III (CIN II/III), especially HPV 16 and 18, cause most cases of human cervical carcinoma. Wide Spectrum HPV Test Kit designed by Triplex International Biosciences Co., LTD. including 28 subtypes,can be used to detect HPV infection sensitively and specifically. Therefore, offering an important method for clinical applications to detect HPV infection. Contents of the Test Kit RT=Room Temperature Volume No. Contents Storage Conditions 500ul 1250ul 1 Wide Spectrum HPV Probe -20 C 500ul 1.25ml 2 Hybridization Enhancer (100x) 4 C 60ml 60ml 3 Mouse Anti-Dig Antibody 4 C 1ml 2.5ml 4 Polymer Enhancer Solution 4 C 1ml 2.5ml 5 Polymerized HRP-Anti Mouse IgG 4 C 1ml 2.5ml 6 DAB Substrate 4 C 1ml 2.5ml 7 DAB Chromogen 4 C 20ul 50ul Present Coverslips(18x18mm) 4 C /RT 20 pieces 50 pieces Humidity Enhancer Solution 4 C /RT 30ml 30ml 1 Manufactory:Triplex International Biosciences Co., LTD Address: Jinshan Science and Technology Incubation Factory 7# Building 4F, Jinzhou North Road NO.7, Jinshan Industrial Park, Foochow Postcode: Tel: Fax: Web: Mail:tib@tibchina.com Free Tel:

6 In Situ Hybridization Wide Spectrum HPV Test Kit Note: For the Wide Spectrum HPV Probe frozen in vials, please centrifugate briefly before opening the vial lid. 1. Materials and Reagents required but not included 1. Heating Appliance (Tibn. M10061) 8. Humidity Chamber 2. Microwave Oven 9. Xylene 3. Incubator 10. Ethanol 4. Light Microscope 11. PBS (PH 7.6) 5. Pipette 12. Haematoxylin 6. Timer 13. ammonia 7. Staining Jar and rack 14. Hydrochloric Ethanol 2. Procedures Reagent Preparation A. Preparation of Reagents in Advance 1. 1x Hybridization Enhancer: Dilute 100x Hybridization Enhancer into 1x Hybridization Enhancer with distilled water, store at room temperature % Humidity Enhancer: Take Humidity Enhancer and distilled water to mix with the proportion of 3:7 to confect 30% Humidity Enhancer. B. Preparation of Reagents Just before Use DAB Solution: Mix DAB Chromogen with DAB Substrate With the proportion of mixture is 1:50 to confect DAB Substrate. The volume of solution should be sufficient to cover the whole area of tissue section. Please stored in the dark before use. Advertisement: Every experiment should append with positive comparison and negative comparison. Operation Process 1. Slide Pretreatment (1.) Prepare paraffin sections 4um onto glass slides coated with APES (2.) Bake the slides at 60 C -70 C for 60 minutes in a dry air oven. (3.) The slides need to be dewaxed and rehydrated. 2. Dewaxing Procedure Solution Times x minutes Fresh xylene 3 x 10 2 Manufactory:Triplex International Biosciences Co., LTD Address: Jinshan Science and Technology Incubation Factory 7# Building 4F, Jinzhou North Road NO.7, Jinshan Industrial Park, Foochow Postcode: Tel: Fax: Web: Mail:tib@tibchina.com Free Tel:

7 In Situ Hybridization Wide Spectrum HPV Test Kit 100% ethanol 1 x 3 95% ethanol 1 x 3 80% ethanol 1 x 3 Distilled Water 1 x 3 Notice: The devaxing time should be prolonged if xylene have been use several several times in order to keep devaxing completely. 3. Microwave procedure: Immerse the slides in 1x Hybridization Enhancer Solution and heat them in microwave oven at high temperature for 30 minutes. (Note: add 1x Hybridization Enhancer Solution at intervals of minutes to make sure sufficient liquid covering the slides.) Set at room temperature for 2-3 minutes. Then wash the slides with distilled water for 1 minutes. 4. Hybridization and Detection: (1.) Add 25ul Wide Spectrum HPV probe to the tissue section, and cover the section with a coverslip (including in the kit). (2.) Denature at 95 C for 5 minutes in the heating appliance. (3.) Incubate the slides at 37 C for 4-16 hours in a humidity chamber with 30% formamide solution. For the samples with low copies of viruses, hybridization overnight is recommended. (4.) Immerse the slides in PBS (heat to 48 C before use) for 1-5 minutes to remove the coverslip, continue to immerse for 5 minutes (slightly shake the container for 2-3 times) (5.) Immerse in PBS (warm up to 48 C before use) for 3x5 minutes (slightly shake the container for 2-3 times). 5. Signal Magnifying and Pigmentation (1.) Tap off and wipe the liquid around the tissue section, add a drop (about 50ul) of Mouse Anti-Dig Antibody, and incubate at 37 C in humidity chamber with distilled water for 30 minutes. (2.) Immerse the slides in PBS for 3x2 minutes at 37 C (slightly shake the container for 2-3 times). (3.) Tap off and wipe the liquid around the tissue section, add a drop (about 50ul) of Polymer enhancer, and incubate at room temperature in humidity chamber with distilled water for 20 minutes. (4.) Immerse the slides in PBS for 3x2 minutes at room temperature (slightly shake the container for 2-3 times). 3 Manufactory:Triplex International Biosciences Co., LTD Address: Jinshan Science and Technology Incubation Factory 7# Building 4F, Jinzhou North Road NO.7, Jinshan Industrial Park, Foochow Postcode: Tel: Fax: Web: Mail:tib@tibchina.com Free Tel:

8 In Situ Hybridization Wide Spectrum HPV Test Kit (5.) Tap off and wipe the liquid around the tissue section, add a drop (50ul) of Polymerized HRP-Anti Mouse IgG, and incubate at room temperature in humidity chamber with distilled water for 30 minutes. (6.) Immerse the slides in PBS for 3x2 minutes at room temperature (slightly shake the container for 2-3 times). (7.) Tap off and wipe the liquid around the tissue section, add a drop (50ul) of DAB solution prepared immediately before use, incubate at room temperature in a humidity chamber with distilled water for 2-10 minutes. Stop the reaction according to microscopic evaluation. (8.) Immerse in PBS for 3x2 minutes at room temperature (slightly shake the container for 2-3 times). (9.) Immerse and wash the slides in distilled water for 1 minutes, pigmentize again with haematoxylin, immerse in HCl for 2 seconds, and thereafter wash in 1% ammonia for 1 minute. (10.) Dehydrate in graded ethanol and seal the slides with neutral balata, Interpret the results under the microscope. Results Positive result: brown precipitation in the nuclei. The punctate signals can be observed in the nucleus when the copies of viruses are low. References 1. Inverse relationship between the expression of the human papilomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia,journal of General Virology (2002),81,1825_ Role of HPV DNA testing in predicting cervical intraepithelial lesions: comparison of HC HPV and ISH HPV, Diagn. Cytopathol. 2003; 29: HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears, J Histochem Cytochem 50: , Electronmicroscopic demonstration of HPV in Oral Warts, Micromologica,13, 27-34,1990 Expiry Date The expiration date is printed on the package (discard after 12 months). Contact us Please check the products immediately when you receive them. Contact us if you find any quality problem.our company will take responsibly for it. 4 Manufactory:Triplex International Biosciences Co., LTD Address: Jinshan Science and Technology Incubation Factory 7# Building 4F, Jinzhou North Road NO.7, Jinshan Industrial Park, Foochow Postcode: Tel: Fax: Web: Mail:tib@tibchina.com Free Tel:

乙型肝炎病毒检测试剂盒 ( 原位杂交 ) 实验时现配试剂 A 1X 蛋白酶 K 工作液取适量 10X 蛋白酶 K, 用蒸馏水稀释 10 倍 (1 份 10X 蛋白酶 K 加 9 份蒸馏水 ), 并将此 1X 蛋白酶 K 工作液存放于室温备用 B DAB 显色液依据需要配制 DAB 显色液, 以可覆盖

乙型肝炎病毒检测试剂盒 ( 原位杂交 ) 实验时现配试剂 A 1X 蛋白酶 K 工作液取适量 10X 蛋白酶 K, 用蒸馏水稀释 10 倍 (1 份 10X 蛋白酶 K 加 9 份蒸馏水 ), 并将此 1X 蛋白酶 K 工作液存放于室温备用 B DAB 显色液依据需要配制 DAB 显色液, 以可覆盖乙型肝炎病毒检测试剂盒 ( 原位杂交 ) 规格 : 500ul 1250ul 编号 : S30322 S30323 ( 操作之前请详细阅读此说明书 ) 检测原理和意义原位杂交技术的原理是采用荧光生物素地高辛等标记的探针, 依据碱基互补配对原理, 与组织切片细胞涂片等标本中的靶序列特异杂交, 经信号放大及显色后, 镜下观察结果此方法具有很高的灵敏性和特异性 HBV( 乙肝病毒 ) 是引起乙型肝炎的病原体,