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.
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