植物组织在逆境胁迫下会产生多种活性氧（ROS），这类物质活性高、稳定性差，通常通过检测其下游产物实现定量与定位。超氧阴离子是重要的活性氧种类（含氧自由基），可将 NBT（氮蓝四唑）还原为不溶于水的蓝色甲瓒产物，从而实现组织中超氧阴离子的原位定位，该方法亦称 NBT 染色法，适用于植物活组织中超氧阴离子的检测。
NBT染色样本保存液（植物用）专用于植物样品超氧阴离子定位染色后的样本保存，可使染色样本常温稳定存放一周，主要适用于幼嫩根尖、叶片等材料的整体染色样本；染色后超氧阴离子聚集区域呈蓝色至深蓝色。本品仅用于科研领域，不用于临床诊断或其他非科研用途。
自备材料
- 新鲜的植物叶片或根、自来水、蒸馏水、95%乙醇
- 超声波、磁力搅拌器、电子天平、滤纸、照相机
- NBT染色液 (植物用)
操作步骤（仅供参考）
1. 样本准备：采集经胁迫（例如重金属）的采集植物幼苗或根尖，自来水稍洗净，置于滤纸上吸干多余水分。
2. 染色：将植物幼苗或根尖浸入NBT染色液中，常温避光浸染2~6 h，至阳性部位出现深蓝色，其余部位为淡蓝色或近无色或呈植物本身的颜色即可。（根据植物幼嫩程度、显色程度调整染色时间）
3. 脱色：用镊子将植物幼苗或者叶片小心取出，浸入蒸馏水中来回漂洗3~5次，置于滤纸上吸干多余水分后，浸入95%乙醇中40℃处理3~16 h，目的是脱去植株幼苗或者叶片本身的叶绿素或者淡蓝色背景，处理期间可多次更换新鲜的95%乙醇。
4. 观察：用镊子取出植株幼苗或者叶片，浸入蒸馏水中来回漂洗3~5次，置于滤纸上吸干多余水分后，将样本转入适量NBT样本保存液中浸泡30 min后，随后可取出拍照。样本可置于该溶液中常温保存一周。
注意事项
1. NBT染色工作液配制好以后需4℃避光保存，一月内使用。存放时间过久，会影响显色。
2. 因任何外在因素都可能刺激植物应激产生超氧阴离子，因此植物样本需要新鲜采集，并尽快完成染色。建议做阴性及阳性空白对照组。
3. 样本染色完成后尽快拍照保存结果。
4. 染色和脱色步骤也可参考如下建议操作：组织放入染液中，抽真空，-0.1 MPa保持负压20~30 min，再于室温下静置染色60 min，弃染色液；加入95%乙醇，于70~80℃水浴锅脱色，每隔10 min换一次95%乙醇，待样品绿色全部褪去后可停止脱色。
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。
7. 推荐NBT染色液货号：N1508542 NBT染色液（0.5mg/mL, pH7.8）；N1508545 NBT染色液（1mg/mL, pH7.8）。
Plant tissues produce various reactive oxygen species (ROS) under stress conditions. These substances are highly reactive and unstable, and are typically quantified and localized by detecting their downstream products. The superoxide anion is a significant type of ROS (oxygen-containing free radical) that can reduce NBT (nitroblue tetrazolium) to an insoluble blue formazan product, thereby achieving in situ localization of superoxide anions in tissues. This method is also known as NBT staining and is suitable for detecting superoxide anions in living plant tissues.
The NBT Staining Sample Preservation Solution (for Plant) is specifically designed for preserving plant samples after localized superoxide anion staining. It allows stained samples to be stored stably at room temperature for one week and is primarily suitable for whole-mount staining samples of materials such as young root tips and leaves. After staining, areas with superoxide anion accumulation appear blue to dark blue. This product is for research use only and is not intended for clinical diagnosis or other non-research purposes.
Materials to Be Prepared by User
- Fresh plant leaves or roots, tap water, distilled water, 95% ethanol
- Ultrasonic cleaner, magnetic stirrer, electronic balance, filter paper, camera
- NBT Staining Solution (for Plant Use)
Procedure (For Reference Only)
Sample Preparation: Collect stressed (e.g., by heavy metals) plant seedlings or root tips. Rinse briefly with tap water and place on filter paper to remove excess moisture.
Staining: Immerse the plant seedlings or root tips in the NBT Staining Solution. Stain at room temperature, protected from light, for 2~6 hours, until positive areas develop a dark blue color, while other areas appear light blue, nearly colorless, or retain the plant's natural color. (Adjust staining time based on plant tenderness and degree of color development.)
Destaining (Chlorophyll/Background Removal): Carefully remove the seedlings or leaves with forceps. Rinse by immersing and agitating in distilled water 3~5 times. Place on filter paper to remove excess water, then immerse in 95% ethanol and treat at 40°C for 3~16 hours. The purpose is to remove chlorophyll or the light blue background from the seedlings or leaves. Replace with fresh 95% ethanol several times during this process.
Observation and Preservation: Remove the seedlings or leaves with forceps. Rinse by immersing and agitating in distilled water 3~5 times. Place on filter paper to remove excess water. Transfer the sample to an adequate amount of NBT Sample Preservation Solution and soak for 30 minutes. Subsequently, the sample can be removed for photography. The sample can be stored in this solution at room temperature for up to one week.
Notes
Once prepared, the NBT staining working solution should be stored at 4°C, protected from light, and used within one month. Prolonged storage may affect color development.
Since any external factors may stimulate stress-induced production of superoxide anions in plants, plant samples should be freshly collected and stained as soon as possible. It is recommended to include negative and positive control groups.
Photograph the stained samples as soon as possible after staining to preserve the results.
For the staining and destaining steps, the following alternative procedure is also recommended: Place the tissue in the staining solution, apply vacuum to -0.1 MPa, maintain the negative pressure for 20~30 minutes, then let it stain at room temperature for 60 minutes. Discard the staining solution. Add 95% ethanol and destain in a water bath at 70~80°C, changing the 95% ethanol every 10 minutes. Destaining can be stopped once the green color of the sample has completely faded.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.
- Recommended NBT Staining Solution Product Numbers:
- N1508542 NBT Staining Solution (0.5mg/mL, pH7.8)
- N1508545 NBT Staining Solution (1mg/mL, pH7.8)
ZJ26F0434134
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