Previous attempts have led to cloudy/opaque solutions. Heat 150 mL of E3 media to 80 degrees Celcius. 15g of Methyl Cellulose is added and stirred for 20 minutes with the heat off with a magnetic stirrer. 350mL of pre-cooled E3 is added to the mix. The solution is then stirred for 3 days at 4 degrees Celcius.
Any modifications to this method or new methods will be appreciated.
Thank you very much.
Dear Kenny,
Attached please find a publication entitled "Zebrafish Development & Genetics " which provides you a good knowledge in the preparation of the solutions indicated in your question. The text below gives an example on how to prepare 2% methyl cellulose solution.
For full documentation, please use the following link:
http://www.mbl.edu/zebrafish/files/2013/03/2013_all-printed-materials.pdf
2% MethylCellulose
2 g Methylcellulose
100 mL of 0.3x Danieau Buffer (i.e., 1.8mM Ca2+).
*Don't heat Methylcellulose as it will become MORE dense (think molecular
gastronomy's hot ice cream);
*Expect to rock this material for 4 days at 4C. Inspect and shake several times;
*Even after it looks dissolved uniformly, there may still be a few undissolved particles which will be visible under microscope and can be disturbing in your images or movies.
Centrifugation of the aliquots in 1.5mL Eppendorf tubes at high speed would help get rid of most of them;
*2% Methylcellulose is very dense and will hold embryos reasonably well for
timelapse, but it will allow you to turn the embryo if needed; Low melting point agarose (Seaplaque cat 50100) can be used to mount embryos
instead.
Hoping this will be helpful,
Rafik
I just stir in the MC powder into VERY COLD, but not frozen, (1-4 c) E3 on ice. I usually use a metal or wooden stick to stir, by hand, for a few minutes. This will make it go *mostly* into solution, with some remaining blobs and bubbles. Then let it sit overnight in the fridge. The next morning you'll have your clean, clear 3% MC!
Preparation of Methyl Cellulose for embedding the fish
Percent 1.7%
1. Heat about 1/3 of the required volume of water to at least 80 °C.
2. Add the methyl cellulose powder to the hot water with agitation using magnetic stirrer .
3. Agitate the mixture until the particles are thoroughly wetted and evenly dispersed.
4. For complete solubilization, the remainder of the water is then added as cold water or ice to lower the temperature of the dispersion. Once the dispersion reaches the temperature at which that particular methyl cellulose product becomes water soluble, the powder begins to hydrate and the viscosity increases. Solution should be cooled to 0-5 °C for 20-40 min.
5. Continue agitation for at least 30 min. after the proper temperature is reached.
6. Keep in the freezer for any small bubble to disappear next day!
I just prepared, it looked difficult in the beginning but it is easier than I expected.
Note: E3 water might make it slightly harder to solubilize the MC, but I prepared it in deionized water just to embed the fish.
Thanks Elizabeth for the protocol! I just made 3% MC and it worked perfectly!
- Badges
- Science method