The aim of this procedure is to culture Xenopus Lavis growth cones for subsequent high resolution image analysis. This is accomplished by first injecting female frogs with hormone to stimulate egg production. The second step is to collect and fertilize the eggs, and then inject them with mRNA or other constructs of interest.
The next day, the embryos are dissected and the neural tubes are isolated, cut up and plated on cover slips. The final step is to image the outgrowing axons and growth cones. Ultimately, high resolution fluorescence microscopy can be used to show changes in protein localization in growth cones over time.
Visual demonstration of this method is critical as the neural tube dissections can be difficult to learn from reading a method section without observing the technique firsthand. Obtain eggs from female frogs previously injected with 400 units of chorionic gonadotropin 12 to 14 hours beforehand. Collect the eggs into one X marks modified ringer solution.
Then fertilize the collected X in vitro with mince testes as previously described, and add 0.1 XMMR after at least 20 minutes. Remove media and incubate the embryos in 2%cysteine in one XMMR at pH 7.8 for three to five minutes to remove the embryo jelly coat. Then pour the embryos into a beaker.
Next, wash the embryos with 0.1 XMMR or 0.1 x modified bath saline three to five times after the final wash. Keep the embryos in 0.1 XMMR at room temperature until injection or if desired. Place the embryos at 14 to 18 degrees Celsius to slow development prior to injection.
Dilute previously prepared capped RNA to a final concentration of 50 to 200 picograms banana liter with double distilled water, and then store on ice until micro injection. Next, prepare the injection needle by pulling a bo silicate capillary using a suter puller or similar instrument. Then under a microscope, use forceps to break the needle tip at an angle to generate a quill like shape and backfill the needle with 0.5 to one microliter of the diluted RNA solution.
Mount the needle on the micro manipulator A medical system's PLI 100 PICO injector is used here. Now, place the fertilized embryos at the one to four cell stage into a plastic dish containing 5%fol in 0.1 XMMR. Calibrate the injection volume for each new micro pipette using a radical or stage micrometer.
Then set the pico injector to deliver the desired amount of RNA for each injection. Here a one nanoliter injection volume is used. Hold the embryos with forceps or if preferred place on a holding platform and inject the desired volume into the animal blasters.
Distribute the injections in several locations throughout the embryo to obtain a uniform distribution of RNA. One to two injections are made in each blaster for a four cell stage embryo, whereas two to four injections are used for a two cell stage embryo. After the injections, transfer the injected embryos to a plastic dish containing 0.1 XMMR and allow them to develop until stage 20 to 23.
Depending on the desired speed of development, the embryos are incubated at a temperature of 14 to 22 degrees Celsius coat PLL treated culture dishes with 10 micrograms per milliliter laminin in PBS and place the plates at 37 degrees Celsius for one hour. Once the incubation time has elapsed, wash the plates three times with PBS being careful not to let the laminin coated surface become exposed to the air after the final wash. Replace the PBS with culture media.
Finally prepare three agros coated dishes by coating the bottom of a cultured dish with 1%aros in 0.1 XMMR and allowing it to harden. Once hardened, fill one dish with steinberg's media variability in the expression of injected mRNA may cause embryos to exhibit a mosaic of fluorescence prior to performing dissections. Screen embryos for the presence of fluorescence and identify those embryos expressing the fluorescent fusion protein in the neural tube.
Then transfer the fluorescent embryos at stage 20 to 23 into the agros coated plastic dish containing Steinberg's Media. Place the dish under a dissecting microscope and use fine forceps to remove the vitelline membrane. Then, while one forceps hold the embryo in place, use the second to make an incision on the side of the embryo and expose the hollow interior.
Next, use both sets of forceps to pinch along the tissue between the dorsal and ventral halves of the embryo to cut the embryo in half and isolate the dorsal portion containing the neural tube. Place the dorsal X explan in a micro centrifuge tube containing two milligrams per milliliter, collagenase in Steinberg's media and place on a rotator for 15 to 20 minutes. Then transfer the dorsal explan to an agro coated dish filled with fresh steinberg's media.
While working under the microscope, dissect the neural tube from the dorsal epidermis and ventral Noor by slipping the tip of the forceps between the epidermis and underlying tissue, and slowly pulling back the epidermis. Then use one forceps to hold the tissue and another to remove tissue surrounding the neural tube. Next, transfer the dissected neural tube to an agros coated dish filled with fresh culture media.
Once several neural tubes have been collected, you sharpen tungsten needles or forceps to cut each tube into around 20 pieces. Then plate the pieces on the laminin coated culture dishes, spreading the eggplants evenly in rows. After plating the neural tubes, the dishes should not be moved as this will disturb the attachment of the cells.
Incubate the plated neural tube explan at 20 to 22 degrees Celsius. A cell culture incubator is not required for the culture of XUS lavis neurons 12 to 24 hours after plating image neurons and growth cones at room temperature. Bear in mind that variable expression of RNA may result in a range of fluorescence levels between growth cones.
This DIC image shows axons and neurites growing outta the X explan at the top left corner of the field of view. The scale bar in this image and the following images represents 10 microns. This higher magnification movie shows the growth cone at the tip of a growing axon.
Finally, this fluorescence microscopy movie shows expression of EB one GFP in a growth cone. While here, we provide the example of imaging, the plus tip fusion protein EB one GFP. This neural explant method can be applied to any number of proteins to elucidate their behaviors within the growth cone during neurite outgrowth.
