植物组织在逆境胁迫下会产生多种活性氧（ROS），因其活性高、稳定性差，通常通过检测其下游产物进行分析。过氧化氢是常见的活性氧之一，在过氧化氢酶催化下，可与 DAB（3,3'- 二氨基联苯胺四盐酸盐）快速反应生成棕红色产物，实现组织中过氧化氢的定位检测，该方法亦称 DAB 染色法，适用于植物活组织的过氧化氢原位染色。
DAB染色样本保存液（植物用）专用于植物样品过氧化氢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；染色后过氧化氢聚集区域呈棕色至深棕色。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
- DAB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）的植物幼苗或根尖，自来水稍洗净，置于滤纸上吸干多余的水分。
2. 染色：将植物幼苗或根尖浸没在DAB染色液中，常温避光染色2~6 h，至阳性部位出现深棕色，其余部位近无色或者呈植物本身的颜色即可。（根据植物幼嫩程度和显色程度调整染色时间）
3. 脱色：用镊子将植株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，脱色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DAB样本保存液中浸泡30 min，随后可取出拍照。样本可置于该保存液中常温保存一周。
注意事项
1. DAB染色工作液配制好以后需4℃避光保存，一周内使用。存放时间过久，会影响显色。
2. 因过氧化氢容易分解，且任何外在因素都可能刺激植物应激产生过氧化氢，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. DAB可能具致癌性，请小心操作，避免直接接触。
5. 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，-0.1MPa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。
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。
8. 推荐DAB染色液货号： D1509200 DAB染色液（1.0 mg/mL, pH3.8, 植物用）；D1509202 DAB染色液（1.0 mg/mL, pH5.5, 植物用）；D1509203 DAB染色液（2.0 mg/mL, pH3.8, 植物用）。
Plant tissues produce various reactive oxygen species (ROS) under stress conditions. Due to their high reactivity and poor stability, ROS are typically analyzed by detecting their downstream products. Hydrogen peroxide is a common ROS. Catalyzed by catalase, it can rapidly react with DAB (3,3'-Diaminobenzidine tetrahydrochloride) to produce a brownish-red product, enabling the localized detection of hydrogen peroxide in tissues. This method is also known as the DAB staining method and is suitable for in situ staining of hydrogen peroxide in living plant tissues.
The DAB Staining Sample Preservation Solution (for Plant) is specifically designed for preserving plant samples after localized hydrogen peroxide 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. Areas with hydrogen peroxide accumulation appear brown to dark brown after staining. This product is for research use only and 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
- DAB 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 DAB Staining Solution. Stain at room temperature, protected from light, for 2~6 hours, until positive areas develop a dark brown color, while other areas remain nearly colorless or retain the plant's natural color. (Adjust staining time based on plant tenderness and degree of color development.)
Destaining (Chlorophyll 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 the chlorophyll 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 DAB Sample Preservation Solution and soak for 30 minutes. Subsequently, the sample can be removed for photography. The sample can be stored in this preservation solution at room temperature for up to one week.
Notes
Once prepared, the DAB staining working solution should be stored at 4°C, protected from light, and used within one week. Prolonged storage may affect color development.
Since hydrogen peroxide is prone to decomposition and any external factors may stimulate its stress-induced production 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.
DAB may be carcinogenic. Handle with care and avoid direct contact.
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 DAB Staining Solution Product Numbers:
D1509200 DAB Staining Solution (1.0 mg/mL, pH 3.8, for Plant Use)
D1509202 DAB Staining Solution (1.0 mg/mL, pH 5.5, for Plant Use)
D1509203 DAB Staining Solution (2.0 mg/mL, pH 3.8, for Plant Use)
ZJ26F0434133
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