计算溶液所需的质量、体积或浓度。
神经HRP示踪显色液 (DAB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,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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。DAB即3,3N-Diaminobenzidine Tertrahydrochloride, 是辣根过氧化物酶的常用底物，在辣根过氧化物酶的催化下，DAB会产生棕色沉淀，该棕色沉淀不溶于水和乙醇，显色后呈棕色，可在显微镜下观察。
神经HRP示踪显色液 (DAB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，该络合物氧化供氢的DAB显色剂，呈棕色，在显微镜下清晰可见。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
| N1508662 | Component | 50T | Storage |
| N1508662A | DAB Assay Buffer | 2×500 mL | RT. Store in the dark. |
| N1508662B | DAB显色液 | 30 mL | -20℃. Store in the dark. |
| N1508662C | DAB增强剂 | 2×1 mL | 2-8℃. Store in the dark. |
| N1508662D | DAB 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%)戊巴比妥钠作为麻醉剂，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 (pH7.4)。
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DAB孵育液：取适量的DAB Assay Buffer和DAB显色液，按DAB Assay Buffer: DAB显色液=39:1的比例混合，即为DAB孵育液，即配即用，不宜保存。
2、配制DAB显色工作液：取适量的DAB孵育液和DAB增强剂，按DAB孵育液: DAB增强剂=2000-8000: 1的比例混合 (具体比例应根据具体时间摸索确定)，即为DAB显色工作液，即配即用，不宜保存。
3、配制1×DAB Wash Buffer：取适量的DAB Wash Buffer (20×)，按DAB Wash Buffer: 蒸馏水=1:19的比例混合，即为1×DAB Wash Buffer；室温保存，6月有效。
4、切片用蒸馏水清洗3次，每次2min。
5、切片入10ml DAB孵育液 (提前20℃温育)，避光孵育20min，其间不断晃动。
6、切片入10ml DAB显色工作液 (提前20℃温育)，避光孵育20min，其间不断晃动。
7、漂洗：取10ml左右的1×DAB 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透明2次，每次2-5min。
10、中性树胶封片，显微镜下观察棕色反应。
注意事项：
1、如果出现高的反应背景或沉淀，表明DAB底物反应过于强烈。
2、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。
3、DAB显色液避免反复冻融，以免显色效率下降。
4、DAB增强剂注意密闭保存，否则显色效率下降。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、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 the neuronal soma, and the outline of neurons can be visualized by histochemical methods. Based on this finding, they developed the HRP neuron tracing technique, namely the HRP method. DAB, short for 3,3'-Diaminobenzidine Tetrahydrochloride, is a common substrate for horseradish peroxidase. Catalyzed by HRP, DAB produces a brown precipitate that is insoluble in water and ethanol, presenting a distinct brown color after chromogenic reaction and being observable under a microscope.
Neuronal HRP Tracing Chromogenic Solution (DAB Method) works on the principle that after animals are anesthetized and injected with HRP, free or complexed HRP reacts with an oxidant to form a complex. This complex then oxidizes the hydrogen-donating DAB chromogenic agent, resulting in a brown product that is clearly visible under a microscope. This reagent is intended for research purposes only and not for clinical diagnosis or any other applications.
| N1508662 | Component | 50T | Storage |
| N1508662A | DAB Assay Buffer | 2×500 mL | RT. Store in the dark. |
| N1508662B | DAB Chromogenic Solution | 30 mL | -20℃. Store in the dark. |
| N1508662C | DAB Enhancer | 2×1 mL | 2-8℃. Store in the dark. |
| N1508662D | DAB Wash Buffer (20×) | 100 mL | RT. |
Operating Procedures (For Reference Only)
(1) Preparatory Work
1. Animal Anesthesia: 3.5% sodium pentobarbital is commonly used as the anesthetic. The anesthetic dose for rats is 0.25-0.35 mL per 100 g body weight.
2. HRP Administration: Methods include pressure injection, electrophoresis, and topical application/injection for the peripheral nervous system.
3. Determine Animal Survival Period: Set the appropriate survival time post-HRP administration according to experimental requirements.
4. Animal Perfusion: After anesthesia, perform intracardiac perfusion fixation via cannulation of the ascending aorta through the left ventricle: First, perfuse rapidly with physiological saline or phosphate-buffered saline (PBS). Next, perfuse with 4% paraformaldehyde fixative, starting at a fast rate then slowing down, with the total duration controlled at 30-40 minutes. Finally, perfuse with 10% sucrose phosphate buffer (pH 7.4).
5. Tissue Harvesting and Sectioning: Immerse harvested tissues in 20% sucrose phosphate buffer. Cut tissues into sections with a thickness of 40 μm and store the sections in sucrose phosphate buffer for later use.
(2) Chromogenic Reaction
1. Prepare DAB Incubation Solution: Mix an appropriate volume of DAB Assay Buffer and DAB Chromogenic Solution at a ratio of 39:1. This solution must be prepared fresh and used immediately; it is not suitable for storage.
2. Prepare DAB Working Chromogenic Solution: Mix the freshly prepared DAB Incubation Solution with DAB Enhancer at a ratio of 2000-8000:1. The optimal ratio should be determined empirically according to specific experimental conditions. This working solution must be prepared fresh and used immediately; storage is not recommended.
3. Prepare 1× DAB Wash Buffer: Dilute the 20× DAB Wash Buffer with distilled water at a ratio of 1:19. The 1× working solution can be stored at room temperature and is valid for 6 months.
4. Wash Sections: Rinse the tissue sections with distilled water 3 times, 2 minutes each time.
5. Incubate with DAB Incubation Solution: Immerse sections in 10 mL of pre-warmed (20℃) DAB Incubation Solution, incubate for 20 minutes in the dark with continuous gentle agitation.
6. Incubate with DAB Working Chromogenic Solution: Transfer sections to 10 mL of pre-warmed (20℃) DAB Working Chromogenic Solution, incubate for 20 minutes in the dark with continuous gentle agitation.
7. Rinse Sections: Rinse sections with approximately 10 mL of 1× DAB Wash Buffer 2-3 times, 5 minutes each time.
8. Mount Sections: Mount sections onto glass slides pre-coated with chrome alum gelatin, then air-dry at room temperature.
9. Dehydration and Clearing: Perform the following steps sequentially:
① Distilled water: 10 seconds.
② 70% ethanol: 10 seconds.
③ 95% ethanol: 10 seconds.
④ 100% ethanol: 2 times, 10 seconds each time.
⑤ Xylene or clearing agent: 2 times, 2-5 minutes each time.
10. Coverslipping and Observation: Seal sections with neutral balsam and observe the brown reaction product under a microscope.
Precautions
1. High background staining or precipitate formation indicates an overly intense DAB substrate reaction; adjust the reaction conditions or reagent ratios accordingly.
2. All labware must be thoroughly clean to avoid contamination with oxidants or reducing agents, which may cause non-specific reactions.
3. Avoid repeated freeze-thaw cycles of the DAB Chromogenic Solution, as this will reduce its chromogenic efficiency.
4. Store the DAB Enhancer in a tightly sealed container; improper sealing will lead to decreased chromogenic activity.
5. For your safety and health, wear a lab coat and disposable gloves during all operations.
6. Use the reagents as soon as possible after opening to prevent 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 Tracing Chromogenic Solution (DAB 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 (DAB Method)
储存条件
2-8°C储存,避光,室温,-20°C储存
运输条件
超低温运输
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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