Methyl Green

Guide (The following is our recommended experimental protocol. This protocol is for reference only and should be modified according to your specific needs).
I. Zebrafish/chicken embryo whole embryo fluorescence staining reference^[1][2]
Steps:
1. Sample fixation: 48 hpf zebrafish embryos or target stage chicken embryos were fixed with 4% paraformaldehyde (HY-Y0333) (prepared with PBS) at 4°C overnight. The fixation time was adjusted according to the thickness of the embryo.
2. Permeabilization: The embryos were washed 3 times with PBS (PBS-T) (HY-K1022) containing 1% Triton X-100 for 5 minutes each time to destroy the egg membrane/tissue barrier.
3. Methyl green staining: dilute 2% methyl green stock solution to 1:5,000-1:10,000 with PBS-T (final concentration of about 2-4 μg/mL), incubate embryos at 4°C for at least 6 hours (overnight for thick embryos), with gentle shaking.
4. Washing and removal of impurities: wash 3 times with PBS, 30 minutes each time, to remove unbound dye.
5. Mounting and imaging: mount the slides with 75% glycerol + 0.1 M Tris·HCl (pH 8.0), use laser confocal microscopy, excitation wavelength 633 nm, emission wavelength 650-750 nm (peak 677 nm).

II. Agarose gel DNA staining^[2]
Steps:
1. Gel preparation: Add 2% Methyl Green (HY-D0163) stock solution to 1% agarose (prepared with TAE buffer) at a ratio of 1:5,000-1:10,000, and cool after gel preparation.
2. Sample loading: Load DNA/RNA ladder (such as 100 bp DNA ladder), conventional electrophoresis (voltage/time adjusted according to the experiment).
3. Fluorescence detection: Use 635 nm red light excitation, 705 nm bandpass filter, exposure for 30-60 seconds, or observe with UV transilluminator (only DNA color development).

III. Flow cytometry to detect cell viability^[2][3]
Steps:
1. Cell treatment: Cultured Raji cells (or PBMCs) were treated with drugs (e.g. 50-100 μg/mL Fludarabine (HY-B0069)) for 24 hours, and then the cell suspension (5×10⁵ cells) was collected.
2. Staining: Add 4 μg/mL Methyl Green (HY-D0163) + 10 μg/mL Propidium Iodide (HY-D0815) and incubate at room temperature in the dark for 15 minutes.
3. Flow cytometry: Use a CyAn flow cytometer, set the excitation wavelength to 633 nm (MG)/488 nm (PI), and the emission wavelength to 677 nm (MG)/609 nm (PI), and analyze the ratio of live and dead cells.

IV. Fluorescence spectroscopy (DNA binding analysis)^[2]
Chinese steps:
1. Solution preparation: Prepare a 100 μg/mL calf thymus DNA (HY-109517) solution in PBS and add methyl green to a final concentration of 10 μg/mL (or 125 ng/mL free state).
2. Spectral detection: Use a fluorescence spectrophotometer, excite at 633 nm, scan the emission spectrum from 645-800 nm, and record the peak at 677 nm (DNA binding state).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.