obtaining cerebellum slices from an embryonic chick brain

Obtaining Cerebellum Slices from an Embryonic Chick Brain

To begin, place fertilized brown chicken eggs into an egg incubator at 38 degrees Celsius until they reach embryonic day 14. On embryonic day 14, cut an opening in the egg to expose the embryo, and decapitate the chick embryo in ovo. Place the head into a Petri dish containing ice-cold PBS.
Under a dissecting microscope, use standard forceps to make an incision behind each eye, all the way through the tissue, and removing the eyes and upper jaw. Then, make a second incision all the way through the pharynx to remove the lower jaw. Next, use standard forceps to remove the skin from the surface of the skull by peeling it away. Then, remove the frontal and parietal bones, revealing the brain.
From a ventral aspect, remove the pharyngeal cartilage and the auxiliary mesenchyme. Then, place the brain, dorsal side up and carefully remove the mesenchyme, dorsal to the hindbrain, taking care not to damage the pia. Next, make an incision all the way through the tissue between the midbrain and the hindbrain to detach the hindbrain, including the cerebellum. Make incisions all the way through the tissue at both the lateral junctions of the cerebellum and the alar plate of the hindbrain. Remove the entire cerebellum, taking care to maintain the integrity of the pia throughout the dissection.
Finally, remove the forming choroid plexus and move the dissected cerebellum into ice-cold HBSS. Transfer the whole cerebellum to the sterile platform of a tissue chopper, using a spatula or a 3-milliliter Pasteur pipette with the tip cut away to widen the aperture. Remove any excess liquid using a pipette.
Set the cutting speed of the tissue chopper to 50% of its maximum value. Then, cut the cerebellum in the required orientation at a thickness of 300 micrometers. Using a 3-milliliter Pasteur pipette, cover the sliced cerebellum in cold HBSS.
Then, transfer the HBSS and the slices to a 60-millimeter Petri dish containing 20 milliliters of ice-cold HBSS, using the 3-milliliter Pasteur pipette with the cut tip. Place the Petri dish under a dissecting microscope, illuminated with a fiber optic light source. Then, use a watchmaker forceps to separate individual slices of brain tissue.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissection of Chicken Embryo Day 14 (e14) Cerebellum
<ol>
	<li>Incubate brown fertilized hens&#39; eggs at 38 &#176;C to embryonic Day 14.</li>
	<li>Using egg scissors, decapitate the chick embryo in ovo and remove the head to a Petri dish containing ice-cold Phosphate buffer saline (PBS) (Figure 1A).</li>
	<li>Using standard forceps, make incisions behind each eye all the way through the tissue, removing the eyes and upper jaw. Make a second incision all the way through the pharynx, removing the lower jaw (Figure 1B).</li>
	<li>Using standard forceps, remove all skin from the surface of a skull by peeling it away (Figure 1C) and remove frontal and parietal bones, revealing the brain.</li>
	<li>From a ventral aspect, remove pharyngeal cartilage and auxiliary mesenchyme.</li>
	<li>From a dorsal aspect, carefully remove the mesenchyme dorsal to the hindbrain, taking care not to damage pia, and separate the entire brain (Figure 1D).</li>
	<li>Make an incision all the way through the tissue between the midbrain and hindbrain and separate the hindbrain, including the cerebellum (Figure 1E).</li>
	<li>Remove the entire cerebellum by making incisions all the way through the tissue at both lateral junctions of the cerebellum and alar plate of the hindbrain, taking care to maintain the integrity of the pia throughout dissection (Figure 1F). Also, remove the forming choroid plexus (Figure 1G).</li>
	<li>Move the dissected cerebellum into ice-cold Hank&#39;s balanced salt solution (HBSS).</li>
</ol>
2. Slice Culture of e14 Cerebellum
<ol>
	<li>Transfer the whole cerebellum to the sterile platform of a Tissue Chopper using a spatula or a 3 ml Pasteur pipette with the tip cut away to widen the aperture. Remove excess liquid using a pipette.</li>
	<li>Cut entire whole cerebellum in the required orientation at 300 &#181;m thickness using the tissue chopper set with a cutting speed at 50% of the maximum. Tissue integrity is most easily preserved in sagittal section; however, orientation is also an important consideration for cell analysis (Purkinje cell dendrites are sagittally aligned, while granule cell axons run transversely, perpendicular to the plane of Purkinje cell dendrites).</li>
	<li>Using a 3 ml Pasteur pipette, cover the sliced cerebellum in ice-cold HBSS.</li>
	<li>Transfer the slices in HBSS to a 60 mm Petri dish containing ice-cold fresh HBSS using a 3 ml Pasteur pipette with a cut tip.</li>
	<li>Under a dissecting microscope illuminated with a fiber optic light source, ensure separation of individual slices using watchmaker forceps. 
	Note: Each dissection, from embryo to slice incubation in culture medium, takes around 10-20 min, depending upon experience. Perform dissections one at a time to ensure that the cerebella spends the minimum amount of time between decapitation and ex vivo culture.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Mickle Laboratory Engineering Ltd</td>
			<td></td>
			<td>Cut at 300&#956;m for best results.</td>
		</tr>
	</tbody>
</table>

Source: Hanzel, M., et al. <a href="https://app.jove.com/v/53421">Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.</a> J. Vis. Exp. (2015).
The video demonstrates the preparation of cerebellum slices. It shows a detailed procedure for isolating the cerebellum from a chick embryo. The slices are then prepared from the dissected cerebellum using a tissue chopper for further experimental analysis.

<img alt="Figure 1" src="/files/ftp_upload/22616/22616_figure 1.jpg" /> 
Figure 1. Dissection of the cerebellum from E14 chick embryos. (A) Decapitate the chick in the egg and remove the head into a petri dish with ice-cold PBS. Remove the lower jaw and the eyes by making an incision behind the eyes and the pharynx (dashed line). (B) Remove the skin from the surface of the skull. (C) Remove the frontal and parietal bones, and (D) remove the brain from the mesenchyme ...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a fertilized hen&#39;s egg at a suitable stage of development.
Cut the shell to access the embryo.
Locate and dissect out the head, then, transfer it to a Petri dish containing chilled phosphate buffer.
Remove the eyes and jaws.
Separate the skin from the surface of the skull.
Next, cut and remove the skull bones.
Clear the surrounding connective tissue to expose the brain.
Separate the forebrain.
Then, detach the cerebellum from the hindbrain and the blood vessel.
Transfer the cerebellum into a chilled salt buffer.
The embryonic cerebellum contains a layer of rapidly dividing neuronal progenitor cells, which makes it valuable for neurogenesis studies.
Place the cerebellum on the platform of a tissue chopper.
Remove excess buffer and slice the cerebellum.
Put chilled salt buffer on the slices.
Transfer the cerebellum slices to a Petri dish containing chilled salt buffer.
Under a microscope, separate individual slices for further analysis.

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Obtaining Cerebellum Slices from an Embryonic Chick Brain

Transcription

Obtaining Cerebellum Slices from an Embryonic Chick Brain