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

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1 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, 常可导致肝硬化肝癌等, 危及患者生命 HBV 原位杂交检测产品通过 HBV 探针与病原核酸的特异结合, 检测组织中 HBV 感染的存在尤其是当组织学上病变轻微不易诊断时,HBV 原位检测常可帮助确诊试剂盒提供的材料和试剂成份保存方法 500ul 提供量 1250ul 1 HBV 杂交液 ul 625ulx2 2 蛋白酶 K(10X) ul 250ul 3 过氧化物酶阻断剂 4 1ml 2.5ml 4 蛋白阻断剂 4 1ml 2.5ml 5 一抗 4 1ml 2.5ml 6 二抗 4 1ml 2.5ml 7 HRP 聚合物 4 1ml 2.5ml 8 DAB 底物 4 1ml 2.5ml 9 DAB 4 20ul 50ul 赠送 18X18mm 盖玻片 4 或室温 20 片 50 片湿盒增效液 4 或室温 30ml 30ml ( 注意 :HBV 杂交液和蛋白酶 K 另小袋包装,-20 保存 ) 用户自备材料和试剂 1. 原位杂交加热器 ( 泰普编号 :M10061) 7. 湿盒 2 个恒温培养箱 8. 二甲苯 3. 光学显微镜 9. 酒精 4. 移液器 10. PBS (PH7.6) 5. 计时器 11. 苏木素 6. 玻片染色缸和染色架 12. 氨水及盐酸酒精实验前需配试剂 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 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. 酶消化处理 : 甩干切片, 将组织周围液体用滤纸吸干, 每张切片滴加适量蛋白酶 K 工作液 ( 约 50 ul, 根据组织大小增减量 ),37 孵育 30 分钟 4. 封闭内源性过氧化物酶 : 蒸馏水冲洗, 小心擦干组织周围液体, 滴加适量过氧化物酶阻断剂 ( 约 50 ul, 根据组织大小增减量 ), 室温水湿盒中孵育 10 分钟 PBS 液洗涤 2 1 分钟后, 蒸馏水洗涤 1 1 分钟, 小心擦干组织周围液体 5. 杂交 : 5.1 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 ); 5.2 放置于 95 原位杂交加热器中变性 5 分钟 ; 5.3 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 ) 6. 蛋白封闭 : 小心擦去组织周围液体, 滴加适量蛋白封闭液 ( 约 50 ul, 根据组织大小增减量 ), 室温水湿盒中孵育 5 分钟 ;PBS 洗涤 1x1 分钟 7. 信号放大与显色 : 7.1 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 ); 7.2 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 ); 7.3 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, 水湿公司名称 : 福建泰普生物科学有限公司 2 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 乙型肝炎病毒检测试剂盒 ( 原位杂交 ) 盒中孵育 30 分钟 ;PBS 室温浸泡 3x2 分钟 ( 轻微振荡容器 ) 7.4 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 ) 7.5 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 : 肝细胞细胞核胞浆或胞膜棕褐色着色其他事项 : 本试剂盒保质期为 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 乙型肝炎病毒检测试剂盒 ( 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 蛋白酶 K 消化 37,30 7 蒸馏水 1 8 过氧化物酶阻断剂室温 10 9 PBS 洗涤 2x1 10 蒸馏水 1 11 加杂交液适量 12 变性 95, % 湿盒增效液的湿盒中孵育 37,4-16h 14 PBS( 用前 48 预温 ) 脱片 + 浸泡 PBS( 用前 48 预温 ) 浸泡 3X5 16 蛋白阻断剂室温 5 17 PBS 洗涤 1 18 加一抗 37,30 19 PBS 浸泡 37,3X2 20 加二抗室温,20 21 PBS 浸泡室温 3X2 22 加 HRP 聚合物室温,30 23 PBS 浸泡室温 3X2 24 DAB 显色室温 PBS 浸泡室温 3X2 26 自来水冲洗室温 1X1 27 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 In Situ Hybridization HBV Test Kit Cat. No. Spec S ul S ul 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. The Hepatitis B virus (HBV) is a mostly double-stranded DNA virus in the Hepadnaviridae family. As one of five hepatitis viruses, HBV causes acute and chronic hepatitis in humans. Despite the current availability of an effective vaccine, almost 1.2 million people worldwide still die each year from HBV related diseases (CDC). In Situ Hybridization HBV test kit can be used to detect HBV infection in tissues, specially when pathological changes can not be diagnosed easily. 1. Contents of the Test Kit RT=Room Temperature Volume No. Contents Storage Conditions 500ul 1.25ml 1 HBV Probe -20 C 500ul 625ulx2 2 Proteinase K (10x) -20 C 100ul 250ul 3 Peroxidase Blocker 4 C 1ml 2.5ml 4 Protein Blocker 4 C 1ml 2.5ml 5 Mouse Anti-Dig Antibody 4 C 1ml 2.5ml 6 Polymer Enhancer Solution 4 C 1ml 2.5ml 7 Polymerized HRP-Anti Mouse IgG 4 C 1ml 2.5ml 8 DAB Substrate 4 C 1ml 2.5ml 9 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 In Situ Hybridization HBV Test Kit 2. Materials and Reagents required but not included 1. Heating Appliance (Tibn. M10061) 7. Humidity Chamber 2. Incubator 8. Xylene 3. Light Microscope 9. Ethanol 4. Pipette 10. PBS (PH 7.6) 5. Timer 11. Haematoxylin 6. Staining Jar and rack 12. ammonia and Hydrochloric Ethanol 3. Procedures Reagent Preparation A. Preparation of Reagents in Advance 1. 30% 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 1. Proteinase K Solution: Dilute Proteinase K (10x) into Proteinase K (1x) to confect Proteinase K Solution and keep in room temperature. 2. 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 % 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 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 In Situ Hybridization HBV Test Kit several times in order to keep devaxing completely. 3. Proteinase K Digestion: Tap off and wipe the liquid around the tissue section, add a drop (about 50ul) of 1 x proteinase K solution, and incubate at 37 C for 30 minutes. 4. Block Internality Peroxidase: wash the slides with distilled water, Tap off and wipe the liquid around the tissue section, add a drop (about 50ul) of peroxidase blocker, and incubate in humidity chamber at room temperature for 10 minutes. Then wash the slides with PBS for 2x1minutes and distilled water for 1 minutes. 5. Hybridization and Detection: (1.) Add 25ul HBV 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). 6. Block Protein: Tap off and wipe the liquid around the tissue section, add a drop (about 50ul) of, protein blocker solution, and incubate in humidity chamber at room temperature for 5minutes;Wash the slides with PBS for 1 minutes. 7. 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). (5.) Tap off and wipe the liquid around the tissue section, add a drop (50ul) of 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 In Situ Hybridization HBV Test Kit 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, cell membrane and cell plasm. 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:

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

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