Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

The overall goal of this experiment is to allow rapid and convenient visualization of different populations of neuronal cells in the developing xenopus embryonic brain. These methods can help answer key questions in the field of primary neurogenesis, such as observing the regulation process of neuronal differentiation in the xenopus central nervous system. The main advantage of this technique is that it allows simultaneous observation of the neural stem-cell progenitor pool, as well as a differentiated primary neural pool within a single experiment.

To begin this procedure, carefully aspirate five to ten embryos out of the glass vial using a plastic or glass pipette without introducing air bubbles. Transfer the embryos into the mounting chamber and observe under a stereoscope. Fill up the mounting chamber with gelatin solution to ensure the rigidity of the section block.

Next, arrange the embryos side-by-side using a pair of fine-tip forceps. Mark the head orientation by drawing an arrow on the rim of the chamber using a cryogenic-compatible marker. For multiple groups of embryos, write down the description of each group on the rim of the corresponding chamber.

Then, carefully place the chamber horizontally in a foam box half-filled with dry ice. Close the lid, and allow the mounting chamber to freeze one at a time for five to ten minutes. After that, proceed with cryosectioning or keep frozen samples at 80 degrees Celsius for at least one to two weeks without losing immunogenicity.

In this procedure, remove the frozen sample block from the chamber by pressing the bottom of the chamber using a blunt stick or a finger. Then, add several drops of tissue-freezing medium onto the sample holding disk. Mount the sample block with the anterior end of the embryos pointing up and allow the mounted block stand inside the cryostat chamber for approximately one minute, or until the tissue-freezing medium becomes opaque.

Immediately install the sample-holding disk onto the microtome with the bottom side of the sample block facing up. Trim off a portion of the sample block using a blade when the sample block is still relatively soft. Then, leave the sample-holding disk on the microtome for at least five minutes to allow its temperature to reach equilibrium.

Afterward, gradually trim the sample block down until the heads of the tadpoles are visible through the translucent gelatin. To increase the trimming speed, increase the section thickness. Monitor the cryostat performance, such as the sharpness and angle of the blade, to insure that subsequent sections are generated in a long strip.

Once the tadpole heads come visible, adjust the settings of the cryostat back to normal, to make sure that the finished slices can form strips and are not overlapping or sticking to the blade. Then, continue trimming until the heads of the tadpoles are almost exposed. Brush off any remnant sections on the stage before beginning to collect the sample sections.

Make approximately 10 to 15 sections and let them form a long strip. Flip over the thick cover glass plate to the side and gently remove the strip from the blade using a fine-tip paintbrush. Next, arrange it on the stage with the long axis parallel to the blade.

Make 10 to 15 sections and let them form a long strip without breaking. It may not be easy and requires practice. One trick is try not to apply the speed too high on the handwheel.

Rather, rotate the handwheel carefully and slowly. After that, pick one positively-charged slide at room temperature and label it with a pencil. Then, press it quickly and firmly onto the strip with the labelled side facing down.

Subsequently, remove the slide from the cryostat chamber. Repeat this step to arrange 20 to 30 slices in parallel on each slide. Air-dry the sides for 10 minutes, then proceed immediately to the immunostaining procedure or store the slides in a slide box at 80 degrees Celsius for up to three to six months before the immunostaining procedure takes place.

Here, we can see the corresponding section planes of the forebrain, midbrain, hindbrain, and spinal cord of a xenopus tadpole which will be used to provide positional references for the following images. The corresponding cross-sections, which were stained with Anti-Sox 3, Anti-Acetylated Tubulin, and DAPI show the relative locations of neural stem-cell pools as well as neurofilaments within the neural tube. And here are the corresponding cross-sections stained with Anti-MyT1 and DAPI which show the relative locations of the differentiated primary neurons within the neural tube.

Once mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it is important to remember to insure sufficient cooling time for the sample block in dry ice. Otherwise, the sample block might not be hard enough and thus difficult to cut.

After its development, this technique paved the way for researchers in the field of developmental neuroscience to explore the neural differentiation in xenopus central nervous systems. After watching this video, you should have a good understanding of how to make sections, and observe specific pools of neuronal cells in a xenopus central nervous system.