甲基绿又称双绿SF，属于碱性染料，是具有金属光泽的绿色微结晶或粉末，分子量为608.78，分子式为C₂₇H₃₅Cl₄N₃Zn。
Methyl Green Staining Solution (1%) 在组织或细胞染色中对细胞核进行染色，常用于鉴定DNA，细胞核中的DNA遇甲基绿会被染成蓝绿色，亦可用于免疫荧光染色或免疫组化染色，10ml染色液可以染色20个样本。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
4%多聚甲醛、蒸馏水、系列乙醇
操作步骤(仅供参考)：
1、样品处理
a) 对于石蜡切片：
二甲苯或脱蜡透明液中脱蜡5 ~ 10min。
更换新鲜的二甲苯或脱蜡透明液，再脱蜡5 ~ 10min。
无水乙醇5min。
90%乙醇2min。
70%乙醇2min。
蒸馏水2min。
b) 对于冰冻切片：
蒸馏水2min。
c) 对于培养细胞：
用4%多聚甲醛固定10min以上。
蒸馏水洗涤2min。
更换新鲜的蒸馏水，再洗涤2min。
2、甲基绿染色
a) 对于上述处理好的样本，用 Methyl Green Staining Solution (1%) 染色5 ~ 10min。
b) 用蒸馏水冲洗2次，此时样本呈蓝色。
c) 95%乙醇处理5s。
d) 用70%乙醇洗涤2次，直接观察或按下列组织切片操作步骤进行。
3、组织切片染色
a) 95%乙醇脱水2min。
b) 更换新鲜的95%乙醇再脱水2min。
c) 二甲苯或脱蜡透明液透明5min。
d) 二甲苯或脱蜡透明液透明5min。
e) 中性树胶或其它封片剂封片。
f) 显微镜下观察，细胞核呈绿色或蓝绿色。
4、荧光染色
a) 如果进行免疫荧光染色，在甲基绿染色液染色后，70%乙醇洗涤2次，每次2min。
b) 95%乙醇脱水2min。
c) PBS或生理盐水或TBS等用于免疫染色或荧光染料染色的溶液浸泡5min。
d) 进行免疫荧光染色或其它荧光染料的染色。
染色结果：细胞核呈绿色或蓝绿色。
注意事项：
1、首次使用本染液时建议先取1~2个样品做预实验。
2、甲基绿染色可以根据染色结果和要求调整时间。
3、为了您的安全和健康，请穿实验服并戴一次性手套操作。
4、试剂开封后请尽快使用，以防影响后续实验效果。
Methyl Green, also known as Ethyl Green SF, is a basic dye. It exists as green microcrystals or powder with a metallic luster, having a molecular weight of 608.78 and a molecular formula of C₂₇H₃₅Cl₄N₃Zn.
The 1% Methyl Green Staining Solution is used for nuclear staining in tissue or cell staining protocols, and is commonly applied for DNA identification. DNA in the cell nucleus will stain bluish-green upon contact with Methyl Green. This solution can also be used for immunofluorescence staining or immunohistochemical staining. 10 mL of the staining solution is sufficient for staining 20 samples.This reagent is for research use only (RUO) and not intended for clinical diagnosis or any other applications.
Materials to Be Prepared by the User:
4% Paraformaldehyde, Distilled Water, Graded Ethanol Series
Operating Procedures (For Reference Only):
1. Sample Preparation
a) For Paraffin Sections:
Dewax in xylene or dewaxing clearing solution for 5–10 minutes.
Replace with fresh xylene or dewaxing clearing solution and dewax again for 5–10 minutes.
Rinse in anhydrous ethanol for 5 minutes.
Rinse in 90% ethanol for 2 minutes.
Rinse in 70% ethanol for 2 minutes.
Rinse in distilled water for 2 minutes.
b) For Frozen Sections:
Rinse in distilled water for 2 minutes.
c) For Cultured Cells:
Fix with 4% paraformaldehyde for 10 minutes or longer.
Rinse in distilled water for 2 minutes.
Replace with fresh distilled water and rinse again for 2 minutes.
2. Methyl Green Staining
a) For the samples prepared as above, stain with 1% Methyl Green Staining Solution for 5–10 minutes.
b) Rinse twice with distilled water; the samples will appear blue at this stage.
c) Treat with 95% ethanol for 5 seconds.
d) Wash twice with 70% ethanol, then observe directly or proceed with the following tissue section staining steps.
3. Tissue Section Staining
a) Dehydrate in 95% ethanol for 2 minutes.
b) Replace with fresh 95% ethanol and dehydrate again for 2 minutes.
c) Clear in xylene or dewaxing clearing solution for 5 minutes.
d) Clear in xylene or dewaxing clearing solution for another 5 minutes.
e) Mount the sections with neutral balsam or other mounting media.
f) Observe under a microscope; the cell nuclei will stain green or bluish-green.
4. Fluorescence Staining
a) If performing immunofluorescence staining, after staining with methyl green staining solution, wash twice with 70% ethanol, 2 minutes per wash.
b) Dehydrate in 95% ethanol for 2 minutes.
c) Immerse in a solution suitable for immunostaining or fluorescent dye staining (e.g., PBS, normal saline, or TBS) for 5 minutes.
d) Perform immunofluorescence staining or staining with other fluorescent dyes.
Staining Results:
The cell nuclei stain green or bluish-green.
Precautions:
1. It is recommended to perform a pilot experiment with 1–2 samples when using this staining solution for the first time.
2. The duration of methyl green staining can be adjusted according to the staining results and requirements.
3. For your safety and health, please wear a lab coat and disposable gloves during operation.
4. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
ZJ26F0130941
危险品化学品经营许可证（带存储）