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.