ニワトリ胚脊髄スライス培養プロトコル

The overall aim of this procedure is to culture chicken embryo slices to maintain normal neuronal development, particularly of spinal motor neurons. This is accomplished by first dissecting the chicken embryo to remove the visceral organs. The second step is to prepare and slice the embryo embedded in an agarro block.

Next, the embryo slice is carefully removed from the agarro. The final step is to transfer the embryo slice into a culture medium, which is incubated for 24 hours at 37 degrees Celsius. Ultimately, immunofluorescence microscopy on thin sections of the embryo slice is used to show the presence and location of spinal motor neurons following the culture period.

So this method can help address key questions in the field of developmental neurobiology. For example, the mechanisms and molecules involved in spinal motor neuron migration. Generally people new to this method will struggle because a lot of care is needed during the dissection stages of the protocol.

Place fertilized 10 eggs with the longest side horizontally in a forced draft incubator at 38 degrees Celsius until they develop to hamburger and Hamilton stage 24. This usually requires approximately four days of incubation. On the second day of incubation, sterilize the eggshell by wiping with a tissue paper soaked in 70%Ethanol carefully pierce the flat end of the egg shell using a 21 gauge needle attached to a five milliliter syringe and remove five milliliters of albumin.

This lowers the embryo away from the shell so that the embryo is not damaged when the shell is opened. Return the eggs to the incubator and incubate them for a further two days. Once the two days have passed, use blunted.

Do more. Number five, forceps to carefully pierce the top middle of the shell and remove around nine centimeter square of shell. Cut around the embryo and carefully lift out the embryo and place in HBSS.

For dissection. All embryos used should be close to stage 24. Any embryos that show any signs of abnormality should be discarded.

Place the dish containing the embryo under a dissection microscope. Use two du al number five forceps to remove the amnion membrane. The chorio Alan to membrane, the Alan Tous and the head next eviscerate the embryo to remove all internal organs by using micros dissecting scissors to cus open the ventral midline of the embryo.

Then hold the embryo around the rostral trunk region with one pair of forceps. Grab the rostral part of the gut and the heart and pull.Quarterly. You should be able to see the embryo somites clearly.

Now trim the thoracic body wall using microdissection scissors. Next, cut the embryo in half transversely between the upper and lower limb buds. Gently remove the lumbar half of the dissected embryo using two forceps to cradle the embryo outta the dissection solution and place in a dry Petri dish using a disposable pasta pipette with about five centimeters.

Cut from the bottom, aspirate approximately two milliliters of molten 4%weight per volume. Aros in PBS pipette approximately 0.5 milliliters of aros on top of the embryo and then suck up the embryo piece into the pipette. Transfer the embryo and aros to a peel away plastic mold without introducing bubbles in the aros.

Gently lower the embryo to the bottom of the aros in the plastic mold with the thoracic section facing downwards and use a 25 gauge needle to ensure that the embryo is straight. The agros will also contain part of the developing limb and it is important that the embryos positioned vertically in the aros to allow this place the plastic mold on ice for around two minutes. To set the aros, take care that the embryo does not change orientation during this period.

Set up the like of VT 1000 S foma similar so that the chamber is filled with HBSS and surrounded by ice. Cold water. Use super glue to fix the aros block to the Vibram platform and then trim the block with a razor blade to leave the embryo piece with one to three millimeters of aros surrounding it.

Start the vibrato and begin slicing the embryo. The slices that include limb bud sections with a clear apical electrodermal ridge are selected and then transferred to a dish of HBSS. Generally, there are only one to two suitable slices per embryo.

Gently remove the agarose around the tissue slices using two needles. It is essential that this is done thoroughly and carefully. Add 500 microliters of culture solution to the required number of wells of a 24 well ultra low attachment tissue culture.

Transfer the slices carefully from the HBSS solution into the well placing two or three slices in each well then place the 24 well plate in the tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide for 24 hours following the culture period at 500 microliters of unbuffered fix to each well of embryo slices and leave on ice for 20 minutes. Then carefully aspirate the total solution and wash three times in PBS for five minute intervals. Then add 30%sucrose in PBS to the wells and place the plate at four degrees Celsius for around six hours or longer until the slices sink.

After the slices have sunk, move the slices to a clean 10 centimeter Petri dish and dab off extra sucrose solution. Then add one milliliter of OCT and allow the slices to equilibrate for five minutes at 20 degrees Celsius. Mount each slice flat in an OCT filled, peel away plastic mold after mounting.

Place the molds vertically on dry ice to solidify mount the OCT blocks onto the cryostat and equilibrate the blocks at minus 24 degrees Celsius for around 20 minutes. Then cut each slice into 15 micron sections and mount on super frosts plus slides. Slides can be stored at minus 80 degrees Celsius until they're needed for immunofluorescent staining as outlined in the text portion of the protocol, this image shows the status of generation of the lateral motor column at hamburger and Hamilton stage 24.

The lateral division of the lateral motor column is visualized by LH X one staining in the left panel. The lateral motor column is visualized by staining with Fox P one in the center panel and the right panel is a merge of the two channels. The midline is shown as a dotted line and DV denotes the dorsal ventral axis here.

The status of the lateral division of the lateral motor column is again shown by LH X one staining in the left panel and the lateral motor column by Fox P one staining in the middle panel with the merged image on the right. These images show development at stage 27. These images show LH X one expression on the left and Fox P one expression in the middle panel in sections of a slice cultured in a medium that supports dissociated cranial motor neuron cell survival.

LH X one is no longer expressed in motor neurons, although there is some survival of LMC cells evidenced by Fox P one expression. DV represents the orientation of the dorsa ventral axis of the spinal cord, and ML represents the orientation of the media lateral axis of the spinal cord lateral motor column. Lateral division cells, again stained for LH X one in the ventral horn in the left panel and the LMC in general stained for Fox P one in the center panel can be observed in slices, cultured in medium containing 4%chick serum and 100 nanograms per milliliter CNTF.

Note that some lateral motor column lateral division cells are found in a lateral position in the ventral horn as indicated by the arrow in the left panel. This image shows status of expression of LH X one and Fox P one in sections of an embryo kept in OVO during the slice culture of the embryo previously shown. This histogram shows the results from quantitation of the percentage of lateral motor column lateral division cells, which stain FOX P one positive and LHX one positive versus lateral motor column cells, which stain FOX P one positive and LHX one.

Negative data is shown for cells found in slice cultures in different conditions compared to control embryos kept in ovo, which represent 100%data were analyzed by using students T-test. The triple asterisk represent a significance level of less than 0.01 Whilst attempting this protocol. It is important to take care when dissecting the embryo and when removing the agros from the slices because it's important to maintain the integrity of the motor axons in the limb bud.