In this video, We are going to show you how to synchronize C elegance at the first level stage. This is a simple method, but very common, so it will be quite useful. You are a beginner still.
If you are a C expert, you may find here has some tips to optimize this protocol. The basis of the synchronization of worms relies on the relative resistance of clen and embryos to alkaline hypochlorite solution rabbit adults. That these adult worms with many embryos inside are incubated with a hypochlorite solution so that they are killed and only embryos remain.
The second part of the synchronization is achieved by the lack of food, so that even though embryos catch at different times, they do not develop farther than L one. The first step to start a blitzing protocol is to have worms at the egg lying stage and there several eggs scattered onto the plate. Use M nine to recover the adults in order to harvest.
Also, the embryos already lead the surface of the plate can be scraped with a soft material such as a piece of an x-ray film wash away the bacteria recovered with several M nine washes. Usually a couple is enough. Once worms have been washed, it's time to add the freshly prepared bleaching solution according to our preferences.
Control the time of incubation while you shake the tube. You can monitor the generation of the adults under the stereo microscope. Once nearly no carcasses are advisable, stop the reaction by adding M nine until the tube is completely full.
To finish the protocol, perform at least three washes. With M nine Eggs are incubated with continuous hesitation overnight in M nine buffer, which allows hatching but prevents further development of the worms. Although synchronization can be performed at any temperature between 15 and 25 degrees, 15 is recommended.
Since development is slower, As We have mentioned during its development, elegan goes through four larval stages prior to adulthood, the time, depending on the temperature at which it is raised. While type C elgan tolerates growth temperatures ranging from 15 to 25 degrees. However, the growth rate is higher.
The upper temperature here, you can see how after 24 hours, the stages at which worms are are different. At 15 and 25 degrees after 48 hours at 25 degrees, we already observe embryos. However, we must wait more than 80 hours to see embryos on the surface of plates.
With worms grown at 15 degrees in order to have optimal results, several issues must be taken into consideration when doing a blitzing experiment. It is also important to recognize worms that are the desire stage for the experiment to perform. So we'll show you how to distinguish the stages either by size, differential interference, contrast microscopy, or that staining.
In this graph, you can appreciate the growing rate of GaN worms, according to the temperature, as we say, are grown being much faster at 25 degrees. There is a correlation between animal size and development in sea elements. With the appropriate software, animals can be measured and then classified into the proper developmental estate.
According to this graph, however, site is a gross marker of developmental stages. More agreed staging can be achieved by observing parts of the seas, anatomy that can be used and recognized either by differential interference, contrast microscopy, or DPI staining. In our lab, we have used this protocol to synchronize worms for different experiments as micro eggs in mono staining, in situ visualization and up is staining.
Moreover, this Protocol also helps you to get rid of contamination. Differential interference contrast or nomar microscopy is used to enhance the contrast. In unstained transparent samples, animals are mounted alive on a base of 2%Agros agros can be prepared previously and stored at four degrees when needed.
It can be melted and kept at 65 degrees. Once the agros is melted, put some tape onto a slide and place them at both sides of a third slide. Carefully take some agros and put a drop on the slide without tape.
Cover the agros with another slide placed transversally. Once the path is solid, separate the two slides by carefully sliding one on the other. The pad will stick to one of them.
Add some of Amazon on the pad to keep worms immobilized. Pick some worms under the dissecting microscope and transfer them to the drop of lele. Avoiding damaging the path.
Take a cover slide and add some nail polish to the edges Carefully place the cover slip on this slide preventing bowel formation. The preparation can be observed immediately under the microscope. One of the easiest features of c Elgan anatomy is Alva.
For example, aava with a Christmas three shape is characteristic of the L four stage. Detailed information of c elgan anatomy through the development can be obtained from www.orla.org. Tapis staining is a common procedure to identify individual cells.
Fixation with the tunnel is a fast method for fixing worms. For dappy staining, first add a drop of M nine buffer to the surface of a microscope slide. Pick some worms directly from the plate and transfer them to the drop of M nine.
Eliminate excess of M nine either with a pipe or with a soft tissue. Add a tunnel to the worms. Do it again.
Once the first drop is dry, add some mounting media with API and the oversleep before taking the preparation to the microscope. The development of the go can be easily observed in warm stent with api.
