The overall goal of this procedure is to examine changes in subcellular localization of proteins upon growth factor signaling. This is accomplished by first injecting animal blasters of xenopus early embryos with RNAs encoding diverse in RFP and the signaling receptor FRIZZLED eight. Next electrodermal implants and or embryo cryo sections are prepared and immuno stained.
Finally, the immuno stain cells are imaged using a fluorescent microscope. Ultimately, results can be obtained showing how specific protein subcellular localization depends on embryonic signaling pathways. As an example, we demonstrate that diverse is recruited from the centrosome to the cell membrane by the FRIZZLED eight receptor.
This method allows researchers to visualize subcellular localization of signaling proteins in vivo using xenopus embryos. This can help address fundamental questions in developmental and cell biology, such as studies of spatial regulation of signaling and cell priority mechanisms. To begin the process of in vitro fertilization, obtain eggs from female frogs that were injected with human chorionic gonadotropin.
12 hours before the experiment, add 0.5 to one milliliter of one X Marx modified ringer solution onto the eggs and fertilize them in vitro with a small piece of dissected testis. After two to three minutes, lower the salt concentration with 0.1 XMMR to activate the motility of sperm. Incubate for 20 minutes.
Upon fertilization, the eggs orient with their animal poles up. Remove the egg jelly coat by replacing the MMR solution with 3%cysteine solution in water for five minutes. Wash the eggs three times with 0.1 XMMR.
Incubate at 13 degrees Celsius to the two to four cell stage. Then transfer the embryos to 3%fi. Call in 0.5 XMMR for micro injection.
Synthesize RNAs from linearized DNA templates using the M message M machine kit and dilute with RNAs free water to a concentration of 0.1 to one microgram per microliter. RNAs for diverse in RFP, the membrane marker GFP CX and FRIZZLED eight are used at 0.1 to one nanogram per injection. Prepare injection needles from capillary tubes using a needle pull and a needle grinder before injection.
Calibrate each needle with water to eject 10 liters of liquid per injection for injections. Place embryos on top of a plastic dish into a large droplet of 3%fial 0.5 XMMR. Draw up one microliter of RNA solution into an injection needle with a narrow shige micro injector.
Inject 10 nanoliters of RNA into each of two animal blasters of two cell embryos. Transfer the injected embryos into a well of a 12. Well plate and incubate at 13 degrees Celsius until they reach the early gastro stage.
Transfer the gastro stage embryos into 0.6 XMMR solution in a six centimeter plastic dish coated with 1%aeros. Manually remove the vitel membrane with forceps. Then use a tsin needle and a hair loop to excise ectodermal explants from the embryos.
Fix them in 3.7%formaldehyde in PBS for 30 minutes. Wash the X explan three times for 10 minutes each in PBS. To stain the nuclei, add davi to the third wash.
Next, mount the X explan on a glass slide by placing them between two strips of scotch tape with the inner un pigmented side of the X explan facing the objective. Add two to three 20 microliter drops of the mounting solution. Place a cover slip on top.
The injected embryos can be cryo sectioned to visualize the distribution of fluorescent proteins in the cell and to coast stain with specific antibodies. At stage 10, fix the embryos for one to two hours at room temperature with dense fixative in a four milliliter glass vial. Next, after washing in PBS, embed the embryos in a 15%fish gelatin, 15%sucrose solution in the same vial.
The next day, transfer the embedded embryos to a plastic mold. Quickly freeze the embedded embryos on dry ice. Then prepare cryo sections using a cryostat.
Perform immunofluorescent staining on the sections, mount slides and image cross sectioned embryos using the same method as described for ectoderm implants. Collect images using a Zes Axio plan fluorescent microscope with the appropriate filters and aome to identify and image specific Z planes within the X explan. In this figure, ersin is localized to the centrosome in electrodermal cells, but trans located to the cell membrane.
In response to Frizzled eight, cex GFP marks the cell membrane in green. Diverse in RFP is in red, and DPI marked nuclei are in blue here. Diverse in RFP is recruited to the cell membrane by Frizzled eight in embryo cryo sections.
Gamma tubulin is a Centro somal marker. After watching this video, you should have a good understanding of the usefulness of XUS embryos and embryonic cells for studies of protein dock authorization and signaling processes at subsidiary resolution.
