The aim of this procedure is to monitor the behavior of the cranial meen chiming culture. Under defined culture conditions. The first step is to collect the uterine horns from a timed pregnant mouse.
8.5 days post coum. Once isolated, the embryos are removed from the decidua and the yolk sac is removed for genotyping. Next, the cranial mechy plants and microdisect and transferred to extracellular matrix coated tissue culture plates.
X explants are allowed to attach and are cultured for up to 48 hours in the presence or absence of defined pharmacological agents. Ultimately, images are taken of explan to show differences in cell migration. This method is useful for answering key questions such as what are the signaling pathways and extracellular matrix proteins involved and controlling the behavior of the cran using kind during neuro fold elevation.
Visual demonstration of this method is critical because the steps of microdissection are difficult to learn and require patience and practice to master To begin if immunohistochemistry will be performed, prepare cover slips otherwise, skip this step and coat tissue culture plates directly with ECM sterilized 12 millimeter circular glass cover slips by dipping them into ethanol. Allow them to air dry. Next place a sterilized cover slip into each well of a 12 well plate.
Working in a laina hood at one milliliter of extracellular matrix or ECM to coat the cover slips or the bottom of a tissue culture plate. Incubate the plate at 37 degrees Celsius for three hours after the incubation, aspirate off the ECM solution and wash three times in PBS. At this point, coated plates may be used immediately or wrapped in and stored at four degrees Celsius for up to two weeks.
Ade in visualizing the translucent embryo. Syl guard plates with resistant black backgrounds are prepared. Following the manufacturer's protocol.
Prepare the dissection tools by inserting a minutian pin into a pin holder. Use a sharpening block to sharpen forceps when everything is prepared. Collect uterus from a timed pregnant mouse that is 8.5 days post quot.
Place the isolated uterus into a dish with sterile PBS and wash. Once under a dissection microscope, separate the individual deciduous using scissors with forceps. Remove uterine connective tissue with a tearing motion, being careful not to create pressure and pop the embryo from the decidua.
Use forceps to carefully tear uterine tissue on the side opposite the placenta. To expose the embryo encased in the yolk sack, use the forceps to pinch the embryo and yolk sack from the placenta and remove the uterine tissue from the dish. Next, using forceps to gently tear, remove the yolk sack from the embryo.
Place the yolk sack in an eppendorf tube for genotyping using a pipette, once isolated stage the embryo by counting the somites. Ideally, the embryo will be between the six and tens, so might stages. At this stage, the neural folds are just beginning to elevate and major morphogenesis of the cranial mesenchyme has not yet occurred.
Once the stage has been determined, transfer the embryo onto a black cell guard plate containing DMEM without phenol red. First under a dissection microscope with the highest possible magnification, focus on the head of the embryo with a dissection needle in each hand, dissect the head anterior to the rmba meres. Next, cut along the midline to bi.
Dissect the head into two equal halves with a dissection needle in each hand. Dissect the outer fringes along the margin of the embryo head and midline. Remove a's needed to prevent confusing desired explan with unwanted tissue.
Explan will contain migrating cranial neural crest, paral mesoderm, and surface ectoderm to remove loosely attached cells. Carefully pipette up and down with a 200 microliter pipette tip. And for Pepin, carefully pick up the explan with a pipette tip.
In approximately 10 microliters of DMEM place in the center of a coated tissue culture, well at 20 microliters of warm DMEM supplemented with 15%FBS, the culture dish can now be transferred to a tissue culture incubator. After approximately one to two hours, the explan is attached and an additional 250 microliters of media can be added. After approximately 24 hours, the explan will be firmly attached to the cover slip.
At this time, pharmacological agents can be added to the media after an additional 24 hours, cells will migrate from thes. Images are typically acquired to document the distance and number of cells that migrate from thes. At this point, sinces begin to show signs of decreased viability with longer incubations seen.
Here are examples of cells migrating from cranial me and chix plants. After 48 hours in different substrates, both the shape and size of cells migrating from the explan are dependent upon. The substrate migration was supported on fibronectin, laminin, max gel, ECM, and hyaluronic acid.
Following this procedure, other methods such as immunofluorescence and live imaging can be used to further probe the pathways that regulate the behavior of the cranial me inkin during neuro fold elevation.
