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神经HRP示踪显色液 (TMB法)
有货
为什么选择此级别
BioReagent,生物染色剂,用于显微镜 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,避光,室温。低温运输 。请查阅批次 COA 获取详细规格。
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SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
上个世纪70年代，Kristensopn和Olsson报道了HRP可神经末梢摄取，经轴浆逆行运输至神经元胞体，经组织化学方法可显示出神经元的轮廓，从而开发出HRP追踪神经元示踪技术，即为HRP法。3,3',5,5'-四甲基联苯胺 (TMB)是非常优越的酶免试验显色剂，能溶解于多种有机溶剂和双蒸水中，为稳定的无色溶液，与适量过氧化脲或双氧水与缓冲液混匀后，与过氧化物酶作用产生清晰的蓝色产物，极易观察，在辣根过氧化物酶的催化下,TMB会产生蓝色沉淀，该沉淀不溶于水和乙醇，显色后呈蓝色，可在显微镜下观察。
神经HRP示踪显色液 (TMB法)是动物经麻醉、注入HRP后，游离或络合型的HRP与氧化剂反应生成络合物，该络合物氧化TMB显色剂，呈蓝色，在显微镜下清晰可见，该检测法较DAB法灵敏。该显色液仅用于科研领域，不宜用于临床诊断或其他用途。
| N1508680 | Component | 50T | Storage |
| N1508680A | TMB Assay Buffer | 500 mL | 2-8℃. Store in the dark. |
| N1508680B | TMB显色液 | 15 mL | 2-8℃. Store in the dark. |
| N1508680C | TMB增强剂 | 2×1 mL | 2-8℃. Store in the dark. |
| N1508680D | TMB Wash Buffer (20×) | 100 mL | RT. |
操作步骤(仅供参考)：
(一)准备工作
1、动物麻醉：多用 (3.5%)戊巴比妥钠、速眠宁或10%水合氯醛等作为麻醉剂，对于戊巴比妥钠大鼠的麻醉剂量为0.25-0.35ml/100g。
2、导入HRP：有压力注射法、电泳法以及周围神经系统的注射涂抹等法。
3、确定动物存活期。
4、动物灌注：麻醉后，经左心室升主动脉插管行心内灌注固定；先用生理盐水或PBS快速灌注；随后用4%的多聚甲醛固定液灌注，先快后慢，时间控制在30-40min；最后用10%蔗糖磷酸缓冲液 (pH7.2-7.4)灌注。
5、取材：取组织置于20%的蔗糖磷酸盐缓冲液中，切片厚度40μm，存于蔗糖磷酸盐缓冲液备用。
(二)显色反应
1、配制TMB孵育液：取适量的TMB Assay Buffer和TMB显色液，按TMB Assay Buffer: TMB显色液=39:1的比例混合，即为TMB孵育液，即配即用，不宜保存。
2、配制1×TMB Wash Buffer：取适量的TMB Wash Buffer (20×)，按TMB Wash Buffer: 蒸馏水=1:19的比例混合，即为1×TMB Wash Buffer，室温保存，6月有效。
3、切片用蒸馏水清洗3次，每次2min。
4、切片入10ml TMB孵育液 (提前20℃温育)，避光孵育20min，其间不断晃动。
5、将切片取出，配制TMB显色工作液：在用过的TMB孵育液中直接加入TMB增强剂，按TMB孵育液：TMB增强剂=2000-8000: 1的比例混合 (具体比例应根据具体实验摸索确定)，即为TMB显色工作液，即配即用，不宜保存。
6、重新将切片浸入刚配置好的TMB显色工作液，20℃温育，避光孵育20min，不断晃动。
7、漂洗：取10ml左右的1×TMB Wash Buffer漂洗切片2-3次，每次5min。
8、贴片，载玻片用铬明矾明胶包被，室温空气干燥。
9、脱水、透明步骤按如下操作：
①蒸馏水: 10s；
②70%乙醇: 10s。
③95%乙醇: 10s。
④100%乙醇 2次，每次10s。
⑤二甲苯或脱蜡透明液2次，每次2-5min。
10、中性树胶封片，显微镜下观察蓝色反应。
注意事项：
1、如果出现高的反应背景或沉淀，表明TMB底物反应过于强烈。
2、TMB显色工作液配置后会出现蓝色絮状颗粒，属于正常现象，使用时需要不断晃动，避免颗粒沉着于切片上，影响后续的观察。
3、所用器皿必须洁净，避免含有氧化剂或还原剂，否则会产生非特异性反应。
4、TMB显色液避免反复冻融，TMB增强剂注意密闭保存，以免显色效率下降。
5、Wash Buffer有挥发性气体，因此使用完要拧紧瓶盖，防止挥发失效。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
7、试剂开封后请尽快使用，以防影响后续实验效果。
In the 1970s, Kristensopn and Olsson reported that horseradish peroxidase (HRP) can be taken up by nerve terminals, transported retrogradely via axoplasm to neuronal somata, and the outlines of neurons can be visualized by histochemical methods. Based on this finding, the HRP neuron tracing technique, namely the HRP method, was developed. 3,3',5,5'-Tetramethylbenzidine (TMB) is an excellent chromogenic reagent for enzyme-linked immunosorbent assays (ELISAs). It is soluble in various organic solvents and double-distilled water, presenting as a stable colorless solution. When mixed with an appropriate amount of urea peroxide or hydrogen peroxide and buffer solution, TMB reacts with peroxidase to produce a distinct blue product that is highly observable. Catalyzed by horseradish peroxidase, TMB generates a blue precipitate that is insoluble in water and ethanol, displaying a blue color after chromogenic reaction for microscopic observation.
Neural HRP Tracing Chromogenic Solution (TMB Method) is designed for the following experimental process: after animals are anesthetized and injected with HRP, free or complexed HRP reacts with an oxidant to form a complex. This complex oxidizes the TMB chromogenic reagent, resulting in a blue color that is clearly visible under a microscope. This detection method is more sensitive than the DAB method.This chromogenic solution is intended for research use only and not for clinical diagnosis or other purposes.
| N1508680 | Component | 50T | Storage |
| N1508680A | TMB Assay Buffer | 500 mL | 2-8℃. Store in the dark. |
| N1508680B | TMB Chromogenic Solution | 15 mL | 2-8℃. Store in the dark. |
| N1508680C | TMB Enhancer | 2×1 mL | 2-8℃. Store in the dark. |
| N1508680D | TMB Wash Buffer (20×) | 100 mL | RT. |
Procedure (For Reference Only)
(1) Pre-Experiment Preparation
1. Animal Anesthesia: Anesthetics such as 3.5% sodium pentobarbital, diazepam, or 10% chloral hydrate are commonly used. For rats, the anesthetic dose of sodium pentobarbital is 0.25-0.35 mL per 100 g of body weight.
2. HRP Administration: Methods include pressure injection, electrophoresis, and topical application in the peripheral nervous system.
3. Determine Animal Survival Time.
4. Animal Perfusion: After anesthesia, perform intracardiac perfusion fixation via cannulation of the ascending aorta from the left ventricle. First, perfuse rapidly with normal saline or PBS.
Subsequently, perfuse with 4% paraformaldehyde fixative at a rapid rate initially and then slow down, with the total perfusion time controlled at 30-40 min. Finally, perfuse with 10% sucrose phosphate buffer (pH 7.2-7.4).
5. Tissue Sampling: Place the harvested tissues in 20% sucrose phosphate buffer. Cut sections at a thickness of 40 μm and store them in sucrose phosphate buffer for later use.
(2) Chromogenic Reaction
1. Prepare TMB Incubation Solution: Mix an appropriate volume of TMB Assay Buffer and TMB Chromogenic Solution at a ratio of 39:1. The TMB incubation solution must be prepared immediately before use and should not be stored.
2. Prepare 1× TMB Wash Buffer: Dilute the 20× TMB Wash Buffer with distilled water at a ratio of 1:19. The 1× working solution can be stored at room temperature for up to 6 months.
3. Wash the sections with distilled water 3 times, 2 min each time.
4. Immerse the sections in 10 mL of pre-warmed TMB incubation solution (20℃). Incubate for 20 min in the dark with continuous shaking.
5. Prepare TMB Chromogenic Working Solution: Remove the sections and directly add TMB Enhancer to the used TMB incubation solution at a ratio of 2000-8000:1 (the optimal ratio should be optimized according to specific experimental conditions). The working solution must be prepared immediately before use and should not be stored.
6. Re-immerse the sections in the freshly prepared TMB chromogenic working solution. Incubate at 20℃ for 20 min in the dark with continuous shaking.
7. Rinse: Rinse the sections with approximately 10 mL of 1× TMB Wash Buffer 2-3 times, 5 min each time.
8. Mount the sections: Use slides pre-coated with chrome alum gelatin. Air-dry the slides at room temperature.
9. Dehydration and Clearing Steps:
① Distilled water: 10 s.
② 70% ethanol: 10 s.
③ 95% ethanol: 10 s.
④ 100% ethanol: 2 times, 10 s each time.
⑤ Xylene or deparaffinization clearing solution: 2 times, 2-5 min each time.
10. Mount the sections with neutral mounting medium and observe the blue reaction product under a microscope.
Precautions
1. High background staining or precipitation indicates an excessively intense TMB substrate reaction.
2. The formation of blue flocculent particles in the TMB chromogenic working solution after preparation is a normal phenomenon. Continuous shaking is required during use to prevent the particles from depositing on the sections and interfering with subsequent observation.
3. All utensils must be thoroughly cleaned to avoid contamination with oxidants or reducing agents, which may cause non-specific reactions.
4. Avoid repeated freeze-thaw cycles of the TMB Chromogenic Solution. The TMB Enhancer should be stored in a sealed container to prevent the reduction of chromogenic efficiency.
5. The Wash Buffer contains volatile components; tightly cap the bottle after use to prevent volatilization and loss of efficacy.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
7. Use the reagents promptly after opening to avoid compromising subsequent experimental results.
规格
别名
神经HRP示踪显色液
英文别名
Neural HRP Tracing Chromogenic Solution | Nerve HRP Tracer Colour Solution(TMB Method)
规格或纯度
BioReagent,用于显微镜,生物染色剂
稳定性与储存
各组分在相应的储存条件下保质期1年。
英文名称
Neural HRP Tracing Chromogenic Solution (TMB Method)
储存条件
2-8°C储存,避光,室温
运输条件
低温运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
技术文档
高级数据
化学和物理性质
敏感性
Light-sensitive
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131449 |
分析证书 | N1508680 |
| ZJ26F0131448 |
分析证书 | N1508680 |
| ZJ26F0131447 |
分析证书 | N1508680 |
| ZJ26F0131446 |
分析证书 | N1508680 |
技术文档和文章
溶液计算器
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