The overall goal of this procedure is to quantify the migration potential of enteric neuro progenitors in the presence of various growth factors. This is accomplished by first dissecting mouse embryonic, small intestines populated by fluorescent neuro crust cells. The second step is to use a vibrating microtome to make 200 micron thick slices of intestines.
Next, these slices are incubated on top of a collagen gel containing growth factors. The final step is to peel off the slices in order to reveal the migrating cells underneath. Ultimately, fluorescence microscopy is used to show the pattern of migrating cells within the collagen gel.
This method can help answer a key question in developmental biology, such as providing a better understanding of the interplay between various signaling pathways underlying the formation of the peripheral nervous system. 12 days after a vaginal plug is found, euthanize the female mouse and remove the uterus, placed the uterus into a glass petri dish filled with ice. Cold PBS cut the uterus transversely between individual deum swellings to isolate each embryo implantation site under a dissecting microscope, use fine forceps to remove the muscle layer of the uterus.
Open the visceral yolk sack and amnion to reveal the embryo. Take care when severing the blood vessels, joining the embryo to the yolk sack as they're intertwined with the developing intestines. Next, insert closed forceps into the abdominal cavity just above the dark red colored liver and allow them to open.
This creates a transversal opening in the abdomen cavity. Pull the opened forceps down towards the posterior end of the embryo to open the abdomen completely. Once opened, grab the connective tissue behind the liver and pull the guts out.
Taking care not to break the intestines. Cut the colon at any location to free the guts from the rest of the embryo. Reserve the tail portion for later.
Tease out the connective tissue from the cecum and then the intestines working carefully not to wound them on the rostral end of the small intestine. Cut away the liver and stomach as well as the meso, nephros and genital ridges if present. Finally, isolate the small intestine by cutting away the cecum and colon as well as the remaining connective tissue.
As before, take care not to nick the intestine. Leave the small intestine in PBS at room temperature for as short a time as possible before embedding. After severing the embryo at the neck, record the number of tail somites to accurately determine the stage of embryonic development.
Before proceeding with embryo dissections, melt 1.5 grams of agros in 100 milliliters of PBS and keep at 50 degrees Celsius. Prepare the embedding mold by cutting a two milliliter micro centrifuge tube lengthwise to excise about one quarter of the tube wall. Pour the melted agros into the mold and let it cool down until it is only slightly warm to the touch.
When ready, hold the intestine with forceps by the folded rostral end and pull it very slowly through the agros, along the length of the mold. This helps to keep the gut straight while the agro sets release the tissue as soon as it begins to resist being moved and keep track of the rostral coddle orientation of the gut. Next, place the mold in a refrigerator for two to three minutes to set completely.
Once set, remove the agros from the mold and trim the excess agros at both ends, making cuts perpendicular to the intestine. Glue the rostral end of the agros block to the metal stage of a microtome and trim the excess aros on all sides. Next, mount the metal stage on the vibrating microtome chamber.
If necessary, adjust the angle of the stage so the intestine is as vertical as possible. Cover the specimen with ice cold PBS before bringing the microtone blade down to a few millimeters under the buffer surface. When ready, make 200 micron transverse cuts of the coddle most small intestine.
Ensuring that each agro slice contains a full intestinal section. Gently deposit the freshly cut slices onto a previously prepared collagen gel. Placing one slice towards the middle of each well to allow the migration of neuro crest cells out out of the eggplants, incubate the slices for three days.
After this time, take the slices off the collagen gel gently with forceps taking care not to damage the gel below. To ensure that the cell migration pattern is not disturbed, it is important to avoid touching the collagen gel directly during the subsequent incubation and wash steps. First, fix the cells with 500 microliters of 4%PFA per well for one hour at room temperature.
Next, replace the fixative with 500 microliters of DPI solution per well and incubate for 10 minutes. At room temperature, carefully remove the DPI solution and wash each well three times with 500 microliters of PBS for five minutes. Lastly, photograph the fluorescent cells embedded in the collagen gel within each well.
In this example, growth factors were used to stimulate the migration of GFP expressing enteric neuro crust cells. The dotted line represents the approximate size and location of the explan. Before it was taken off the gel, the number and spread of GFP expressing cells migrating out of intestinal explan was found to be significantly greater in treated versus untreated conditions.
After watching this video, you should have a good understanding of how to determine the migration potential of enteric neural progenitors by incubating intestinal slices over a collagen gel containing growth factors.
