Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

In this video, we will show how the very common histological stain methyl green can be used as an extremely convenient nuclear label in fluorescent microscopy with the special applications in laser confocal microscopy of thick biological specimens such as whole embryos. After shortly introducing methyl green as a DNA specific stain, we will describe the method for preparing stock and working solutions for fluorescent staining. We will then show the experimental procedure to achieve nuclear staining and hold several fish embryos and examples of confocal images obtained.

Finally, methyl greens excellent physical chemical properties will be demonstrated and discussed. Freddy Misha discovered DNA in 1869 and showed a few years later that it formed precipitates with a relatively new annal and stain methyl green. Methyl green has two positive charges that allow it to strongly interact with DNA at its major group preferentially binding to at reach regions.

The most extended use of methyl green has been its combination in an nasal solution with RNA stain perine, a methyl developed by UNA and Poppen Heim at the beginning of the 20th century. Very little has been analyzed, however, on fluorescent properties of methyl green, and this is why we decided to test for the possibility that it could be used as a very high affinity and specific fluorescent label. For DNA on cells.

Methyl green is a al methane stain with seven methyl roots. This seventh group is easily lost and it di reverts to crystal violet. For this reason, there is a arrival amount of crystal violet mixed with methyl green to remove crystal.

Violet methyl green solution may be washed with chloroform to extract crystal violet. As we will show immediately for this purpose, we prepare 4%ACU solution under a fume hood. We mix it with at least two parts of chloroform and centrifuge to accelerate phase separation.

After centrification, we obtain an aqueous phase with methyl green and a lower organic phase with chloroform and crystal violet. We recovered the upper phase and repeated until the lower phase appears clear. At the end, the upper phase is recovered and diluted.

This is solution is a stable on the work bench for months. Now we will show how to proceed to tend whole MO in embryos layer with meth green. For confocal microing, we'll use the high transplants fish embryo.

As an example, Obtain zebrafish embryos and wash the debris away with a strainer. Incubate them to the desired stage in fish tank water or embryo medium. Remove the corium with fine tweezers, fix the embryos in 4%for formaldehyde for this few hours or overnight.

Then wash the embryos in PBST for several times. Prepare a methyl green working solution by diluting the stock solution five to 10, 000 times in PBST. Incubate the embryos in the solution for at least three hours at room temperature or overnight of four decreased Celsius.

In this example, confocal images will be obtain in a lesser confocal microscope ated with spectral detectors. Maximal excitation is achieved with a 633 nanometer laser and the detector is set to a wide range. In the red area of the visible spectrum.

Methyl green red emission makes it possible to get good quality nuclear imaging even from deep specimens like whole embryos with a minimal signal loss. This first series shows a zebrafish retina whose nuclei has been stained with a well-established DNA stain to pro three. After one minute we zoom out.

An ABL scan region becomes evident when we perform the same experiment with methyl green, no evident bleaching is seen. In the following series, we show representative results of what can be obtained using the protocol described here, mitotic cell from zebrafish epidermis in which mitotic figures can be observed. IIC nucleus from the same specimen where nuclear architecture can be seen.

3D reconstruction of a part of a zebrafish larvae epidermal cell labeled with methyl green amalin, a more panoramic view of different nuclear shapes within a 48 hours port fertilization, silverfish, br retina. We have shown here an easy way of preparing an extremely inexpensive fluorescent DNA stain that can be used in nuclear label, Although we just showing as an example, the of hold several. This technique can apply to your embryos or tissues, either whole mount or sectional.

It can also be used for DNA staining in gels and for DNA determination in solution using photometry.