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(PP-PLM-A-002) Revision: 1.

5 Date: 2015/5/1

1 E En ng gl li is sh h M Mo ou us se e A An nt ti i- -H Hu um ma an n Actinin Alpha

4 M Mo on no oc cl lo on na al l A An nt ti ib bo od dy y ( (A An nt ti i- -A AC CT TN N4

4 m mA Ab b) ) C Ca at ta al lo og g I ID D: : D DH H0

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03 3 For in Vitro Diagnostic Use (IVD): USA, EU and Taiwan For Research Use Only (RUO): Other countries For Professional Users Intended Use Mouse Anti-Human Actinin, Alpha 4, clone 13G9 is intended for the semi-quantitative detection of Actinin, Alpha

4 protein in paraffin sections. The clinical interpretation of any positive staining or its absence should be complemented by morphological studies and histology studies with proper controls. Evaluations should be made within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist. Principles of Procedure Immunohistochemical(IHC) staining techniques allow for visualization of tissue constituents localization under microscope through a two step process including antigen-primary antibody interactions and the detection of bound antibody by a chromogen. Reagent Provided 1.

100 ug of monoclonal mouse antibody, clone 13G9, to Actinin Alpha 4, diluted in 1X PBS, pH 7.2. Reagent Required but Not Provided 1. Blocking solution or Antibody Diluent (DAKO, Cat # S3022) 2. Immunodetection Kit- EnVision Detection Kit, Peroxidase/DAB,Rabbit/Mouse (DAKO, Cat # K5007) Users are advised to use the reagents recommended by Abnova, otherwise the expected results may not be achievable. Isotype IgG2b Immunogen peptide, 'NQSYQYGPSSAGNGA'. Specificity Human Actinin, Alpha

4 (PP-PLM-A-002) Revision: 1.5 Date: 2015/5/1

2 Antibody Concentration Refer to vial label for batch specific Ig concentration. Recommendation on Working Condition Suggest primary antibody concentration: 1.5ug/ml overnight at 4°C, and do not re-use. Heat mediated antigen retrieval using 0.01M citrate retrival solution (pH 6.0) is recommended. These are guidelines only and the users should determine their own optimal condition. Storage and Stability Please refer to vial label for expiration date and store at -20°C. Do aliquot to avoid repeated freezing and thawing. Do not mix different lot of antibody into one and not use after expiration date. Storage condition other than those specified in the package insert, they must be verified by the user. Specimen Preparation Tissues specimens preserved for IHC staining by formalin fixation. Staining interpretation The cellular staining pattern for anti-Human Actinin, Alpha

4 is cytoplasm or nucleus. Staining Procedure The following protocol is used in ABNOVA. Customer can refer to the protocol from the detection system selected. Deparaffinize sections and rehydrate using 1X PBS Pre-treat the sample with the following procedure: Place sample in 1X citrate buffer (pH 6.0) in pressure cooker under 125℃ for 4min and under 90℃ for 45min, cool sample subsequently. Wash sample with 1XPBS. Step-by-step procedure: 1. Incubate sections in 3% H2O2 in 1X PBS at room temperature for

10 minutes and then wash the sections with 1X PBS. 2. Incubate sections in blocking solution for

10 minutes. 3. Add primary antibodies (1.5 ug/ml) and incubate the sections overnight at 4°C, wash sample with 1X PBS afterwards. 4. Incubate sections with labeled polymer(1) (HRP-conjugated

2 Ab) for

30 min followed by washing the sections (PP-PLM-A-002) Revision: 1.5 Date: 2015/5/1

3 with 1X PBS. 5. Application of chromogen and substrate solution(2) (DAB or other suitable peroxidase substrate). Wash sample thoroughly under running tap water. 6. Counter stain the samples in Mayer's hematoxylin. 7. Dehydrate and mount samples. Note: (1) (2) These are the components of the Immunodetection Kit- EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DAKO, Cat # K5007)) mdi Europa GmbH Langenhagener St. 71, D-30855 Langenhagen, Germany (PP-PLM-A-002) Revision: 1.5 Date: 2015/5/1

4 繁 繁体 体中 中文 文 卫署医器制壹字第004236号亚亚诺 诺法 法α α- -辅 辅肌 肌动 动蛋 蛋白 白44单单株 株抗 抗体 体( (未 未灭 灭菌 菌) ) A Ab bn no ov va a A An nt ti i- -A AC CT TN N4

4 m mA Ab b ( (N No on n- -s st te er ri il le e) ) 目 目录 录号 号码 码: : D DH H0

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03 3 体外诊断试剂(IVD):美国、欧盟及台湾 限研究用(RUO):其他国家 本产品限专业人员操作使用 用途 α α- -辅 辅肌 肌动 动蛋 蛋白 白44的的老 老鼠 鼠单 单株 株抗 抗体 体, ,c cl lo on ne e

1 13 3G G9 9, ,适 适用 用於 於半 半定 定量 量测 测定 定( (semi-quantitative detection) )经 经 马 马林 林固 固定 定、 、石蜡包 埋的人类组织切片中的α α- -辅 辅肌 肌动 动蛋 蛋白 白44. .临 临床 床上 上任 任何 何阳 阳性 性或 或阴 阴性 性的 的染 染色 色结 结果 果之 之判 判定 定需 需辅 辅以 以形 形态 态学 学( (m mo or rp ph ho ol lo og gy y) )和 和组 组织 织学 学((h hi is st to ol lo og gy y) )的 的判 判读 读结 结果 果为 为辅 辅助 助判 判断 断, ,并 并且 且要 要有 有适 适当 当的 的对 对照 照组 组. .结 结果 果的 的判 判读 读需 需由 由合 合格 格的 的病 病理 理学 学家 家来 来执 执行 行, ,并 并依 依病 病人 人的 的病 病历 历及及其 其他 他的 的诊 诊断 断测 测试 试( (d di ia ag gn no os st ti ic c t te es st ts s) )结 结果 果来 来得 得出 出最 最终 终的 的结 结论 论. . 实验原理 免疫组织化学染色(IHC)技术可以让我们透过显微镜观察到组织里特定成份(通常是蛋白)的位置.此技术共有

2 个实验步 骤,第一个步骤为抗原和一级抗体的结合(antigen-primary antibody interactions),第2步则为以色原体(chromogen)侦 测该结合的抗体,使该抗原-抗体的结合物在组织里呈色,而显现出其所在的位置. 提供的试剂 1.

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00 0 u ug g 的的α α- -辅 辅肌 肌动 动蛋 蛋白 白44老老鼠 鼠单 单株 株抗 抗体 体, ,c cl lo on ne e

1 13 3G G9 9, ,溶 溶於 於11x x PBS, pH 7.2. 需要但未提供的试剂 1. Blocking solution or Antibody Diluent (DAKO, Cat # S3022) 2. Immunodetection Kit- EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DAKO, Cat # K5007) 使用者若未搭配使用亚诺法建议之试剂,可能会无法达到预期的实验结果. Isotype IgG2b 免疫原(Immunogen) 其免疫原是一段序列为 NQSYQYGPSSAGNGA 的多胜肽. 专一性 办认人类α α- -辅 辅肌 肌动 动蛋 蛋白 白44. (PP-PLM-A-002) Revision: 1.5 Date: 2015/5/1

5 抗体浓度 请参考产品试剂管上的标签资讯. 建议的实验条件 建议的抗体浓度为 1.5 ug/ml,在4°C 作用隔夜(overnight),请勿重复使用.以热处理的方式进行抗原恢复(antigen retrieval),建议使用 0.01M citrate retrival solution (pH 6.0).这里提供的仅是建议,客户可依髯缘那榭龅髡鲎罴 的实验条件. 保存期限及保存条件 存放於-20°C,产品的保存期限标记於产品试剂管的标签上.使用前请先进行分装,每次使用时只取出所需使用抗体, 以避免或减少因反覆冷冻和解冻而影响抗体的品质.不同批号的抗体请勿混合使用,如果已经过了保存期限也请勿再使 用.其他未载明於本说明书里的保存条件,使用者须自行确认其保存效果. 样本的制备 要用於 IHC 染色的组织样本需先用马林固定. 理想的染色结果 此抗体的染色型态为细胞质或细胞核. 染色步骤 以下的实验步骤(protocol)是亚诺法所使用的.客户亦可参考其所选用的侦测系统(detection system)所提供的实验步骤. 组织切片的去蜡以及使用 1X PBS 复水(rehydrate) 依以下流程进行样本的前处理: 将样本(组织切片)放置在 1X citrate buffer (pH 6.0)中,置於 125°C 的压力锅 (pressure cooker)里4分钟,之后再置於 90°C 的压力锅 (pressure cooker)里45 分钟,然后冷却样本.用1X PBS 清洗组织切片. 实验步骤 1. 将组织切片浸泡在含3% H2O2的1X PBS中,置於室温(RT)10分钟,然后以1X PBS清洗组织切片. 2. 将组织切片浸泡在blocking solution中10分钟. 3. 加入α α- -辅 辅肌 肌动 动蛋 蛋白 白一级抗体 (1.5 ug/ml)然后将组织切片放置在4°C 隔夜(overnight),之后以1X PBS清洗组织切 片. 4. 加入labeled polymer(1) (HRP-二级抗体)并作用30分钟,之后以1X PBS清洗组织切片. 5. 加入色原体及substrate solution(2) (DAB or other suitable peroxidase substrate).并在流动的自来水下清洗组织 切片. 6. 将组织切片用Mayer's hematoxylin染剂进行对比染色(Counter stain). 7. 将组织切片进行脱水之后使用封片胶进行封片. (PP-PLM-A-002) Revision: 1.5 Date: 2015/5/1

6 备注: (1)(2) 这些是Immunodection Kit- EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DAKO, Cat # K5007)里 的试剂. mdi Europa GmbH Langenhagener St. 71, D-30855 Langenhagen, Germany