本稀释液为即用型工作液，专门用于免疫组织化学（IHC）、免疫细胞化学(ICC)等免疫实验中一抗与二抗的稀释。其配方含有稳定剂与保护成分，能维持稀释后抗体的活性，能加速一抗和抗原的结合，节约很多时间。使用本产品稀释的抗体工作液可在4°C保存并重复使用不少于一周，有助于节约宝贵的抗体试剂。
1小时替代过夜，一步孵育，无需封闭。兼容：该稀释液也适用于4度过夜孵育，一抗/二抗通用。
主要成分：专有缓冲体系、蛋白稳定剂、防腐剂。
免疫组化实验步骤（参考）：
| 步骤 | 操作
| 关键细节与建议
| 脱蜡与水化
| 切片经二甲苯脱蜡，梯度乙醇水化至蒸馏水。
| 确保彻底脱蜡，否则易导致染色不均或背景过高。
| 抗原修复
| 根据一抗要求，采用适当的缓冲液（如枸橼酸盐（货号C752119）或EDTA缓冲液（货号：E745530））进行热诱导抗原修复。
| 修复后需自然冷却至室温，再进行后续步骤。
| 阻断内源性酶
| 使用内源性过氧化物酶阻断液（货号：E752115）溶液孵育10-30分钟，以消除内源性过氧化物酶活性。
| / | 一抗孵育
| 用本稀释液按抗体说明书推荐起始浓度配制一抗工作液，滴加至切片。
| 孵育方案二选一：
A.快速法：37°C孵育1-2小时，一定防止干片。
B.常规法：4°C过夜孵育，随后用TBST充分洗涤3次
| 二抗孵育
| 用本稀释液将HRP标记的二抗按1:200至1:500稀释，滴加至切片。
| 室温或37°C孵育30分钟至1小时。随后用TBST充分洗涤3次。
| 显色与复染
| 滴加DAB（货号：D405772）等显色底物，在显微镜下控制显色时间。
| DAB显色时间需灵活掌握，至目标信号清晰而背景较浅时即可终止。
| 细胞核染色
| 自来水终止后， 用苏木素（货号：H1507743）进行细胞核复染，染色3-5分钟。
| 如果蛋白是细胞核表达，可以不染苏木素或者减少染色时间。
| 封片
| 切片经脱水、透明后，使用无毒性中性树胶（货号：A1508972）封片，长期保存。
| 常规中性树胶含有二甲苯，建议用无毒性中性树胶。 |
|
孵育时间选择：您可以根据蛋白的表达量，做适当的调节：
方案
| 一抗孵育
| 二抗孵育
| 适用场景
| 方案一
| 37°C，1-2小时
| 室温/37°C，30分钟-1小时
| 高丰度蛋白，非特异性结合比较强，需当天出结果。
| 方案二
| 4°C，过夜
| 室温/37°C，30分钟-1小时
| 检测低丰度靶标，追求高信噪比。
|
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注意事项：
1、使用范围：本产品仅限于专业人员的科学研究用途，不得用于临床诊断、治疗或任何食品、药品领域。
2、抗体回收：为延长回收后抗体的使用寿命，建议在4°C条件下进行一抗孵育。
3、核心操作要点说明。
4、抗体稀释：始终以抗体生产商说明书推荐的起始浓度作为一抗稀释依据。二抗的通用起始稀释范围在1:200至1:500。
This diluent is a ready-to-use working solution, specially formulated for diluting primary and secondary antibodies in immunoassays including immunohistochemistry (IHC) and immunocytochemistry (ICC). It contains stabilizers and protective ingredients to maintain the activity of diluted antibodies, accelerate the binding between primary antibodies and antigens, and significantly save experimental time. Antibody working solutions prepared with this diluent can be stored at 4°C and reused for no less than one week, helping conserve valuable antibody reagents.
Replace overnight incubation with just 1 hour; one-step incubation with no blocking required.
Compatibility: This diluent is also suitable for 4°C overnight incubation and is universally applicable to both primary and secondary antibodies.
Main Ingredients: Proprietary buffer system, protein stabilizer, preservative.
Reference Immunohistochemistry (IHC) Experimental Procedures:
Step
| Operation
| Key Details and Recommendations
| Deparaffinization and Hydration
| Deparaffinize sections with xylene, followed by gradient ethanol hydration to distilled water.
| Ensure complete deparaffinization; otherwise, uneven staining or high background is likely to occur.
| Antigen Retrieval
| Perform heat-induced antigen retrieval with an appropriate buffer (e.g., Citrate Buffer, Cat.No. C752119 or EDTA Buffer, Cat.No. E745530) according to the requirements of the primary antibody.
| Allow the sections to cool naturally to room temperature after retrieval before proceeding to the next steps.
| Endogenous Enzyme Blocking
| Incubate sections with Endogenous Peroxidase Blocking Solution (Cat.No. E752115) for 10–30 minutes to eliminate endogenous peroxidase activity.
| / | Primary Antibody Incubation
| Prepare the primary antibody working solution with this diluent at the initial concentration recommended in the antibody instructions, then add dropwise to the tissue section.
| Choose one of the two incubation protocols:A. Rapid protocol: Incubate at 37°C for 1–2 hours; avoid drying out of sections at all times.B. Conventional protocol: Incubate overnight at 4°C, then wash thoroughly 3 times with TBST.
| Secondary Antibody Incubation
| Dilute HRP-conjugated secondary antibody at a ratio of 1:200 to 1:500 using this diluent, and apply dropwise to the sections.
| Incubate at room temperature or 37°C for 30 minutes to 1 hour. Afterwards, wash thoroughly 3 times with TBST.
| Color Development & Counterstaining
| Add DAB (Cat. No.: D405772) or other chromogenic substrates, and control the color development time under a microscope.
| The DAB color development time should be flexibly controlled. Terminate the reaction once the target signal becomes clear with low background staining.
| Nuclear Staining
| Terminate the reaction with tap water, then perform nuclear counterstaining with hematoxylin (Cat.No.: H1507743) for 3–5 minutes.
| If the target protein is expressed in the cell nucleus, hematoxylin staining can be omitted or the staining time can be shortened appropriately.
| Mounting
| After dehydration and clearing of the sections, mount slides with non-toxic neutral balsam (Cat.No.: A1508972) for long-term preservation.
| Conventional neutral balsam contains xylene; the use of non-toxic neutral balsam is recommended. |
|
Incubation Time Selection
You can appropriately adjust the incubation time according to the expression level of the target protein:
Protocol
| Primary Antibody Incubation
| Secondary Antibody Incubation
| Application Scenario
| Protocol 1
| 37°C, 1–2 h
| Room temperature / 37°C, 30 min–1 h
| High-abundance proteins with relatively strong non-specific binding; results required on the same day.
| Protocol 2
| 4°C, Overnight
| Room temperature / 37°C, 30 min–1 h
| Detection of low-abundance targets; pursuit of high signal-to-noise ratio.
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Precautions:
1. Scope of application: This product is only for scientific research use by professionals. It shall not be used for clinical diagnosis, treatment, or any food and pharmaceutical applications.
2. Antibody recovery: To prolong the service life of recovered antibodies, it is recommended to perform primary antibody incubation at 4°C.
3. Key operational notes.
4. Antibody dilution: Always follow the starting dilution concentration recommended in the antibody manufacturer’s instructions for primary antibody preparation. The general starting dilution range for secondary antibodies is 1:200 to 1:500.
ZJ26F0535740
危险品化学品经营许可证（带存储）