脳室下帯恩顔：Wholemount染色と上衣の流れ

Hi, I'm Zaman. I am Arturo Alvarez bia. We're here in Arturo's Lab today, part of the Department of Neurosurgery and the Eli Edith Broad Center for Regeneration Medicine and stem Cell research, and we're gonna show you how we prepare whole mounts of the lateral ventricle walls.

These whole mounts can be used either fixed for immuno staining or as live preparations to analyze the penal flow. The whole mount technique that Mann is going to illustrate to you today was developed over the years in the lab and perfected to study the whole wall of the lateral ventricle, including the penal cells and the underlying germinal activity in the subventricular zone where the neural stem cells lie. It was originally developed by Fiona Dutch and then perfected by HIE Victor and Casu Moto.

All of them are no longer in the lab. The following diagrams illustrate how to prepare a whole mount of the lateral wall of the lateral ventricle. The brain is first divided into two hemispheres.

A coronal cut is made posteriorly to reveal the hippocampus and cross-section shown here in purple. The medial wall, which covers the hippocampus and is shown in purple, is then peeled away from the lateral wall, which covers the stray aum and is shown in pink. The two walls will be spread away from each other like opening a book, and the medial will be cut away leaving the lateral wall exposed.

The first section of this video addresses the whole mount dissection, but this may not be the first step. Chronologically, if plans include analyzing a penal flow, please refer to the written protocol for details Before each dissection, make sure all tools are sterile and at hand. Put a six centimeter dissecting dish with 37 degrees Celsius, L 15 leitz medium under a fluorescent dissecting stereo microscope, and place a freshly dissected mouse brain into the dish under the stereo microscope.

Use the fine forceps to dissect away the olfactory bulbs. Use a stab knife to divide the brain into its two hemispheres. Cut coronal at the posterior end of one hemisphere.

To expose the cordal hippocampus and cross-section, insert the stab knife into the small ventricular space between the cortex and hippocampus, and cut to release the cortex where it connects dorsally to the hippocampus. Cut off a wedge of cortex at the corner and use fine forceps to slowly peel it away from the hippocampus. Continue to peel away the cortex as it wraps around to the ventral edge of the hippocampus.

Use a stab knife to release the hippocampus from the ventral parts of the cortex. Use the knife and fine forceps to retract the hippocampus away from the cortex. Carefully proceeding anteriorly without damaging the ventricle walls.

The two walls should open away from each other. Like a book with the knife, cut away the parts of the cortex that overhangs the lateral wall. To better visualize this surface as resistance to the retraction increases, two additional cuts will have to be made.

One dorsally where the medial and lateral walls meet, and a similar cut ventrally. Use small strokes of the stab knife to make these cuts. These additional cuts allow the medial wall to be further retracted to reveal the anterior of the ventricle.

Adjust the lighting such that the shadows reveal the junction between the medial and lateral walls. As a valley between the two walls. Use the knife to cut exactly in the valley, remove the thalamus and any overhanging cortex.

The hole mount should be used immediately for a penal flow analysis. If dissecting hole mounts for immuno staining, many hole mounts can be dissected at one sitting if they're immersed in fixative as they are completed. The next section of the video will demonstrate how to perform assays of fluid flow generated by ependymal cell Celia.

Using fluorescent microbeads. The freshly dissected lateral wall hole melt is first secured to the dissection dish with two insect pins. The tip of a glass micro pipette filled with fluorescent microbeads is then lowered onto the surface of the whole mount, and fluorescent beads are gently deposited onto the surface while time-lapse.

Video records the movement of the beads for the append flow assay. A glass micro pipette should be prepared before the whole mount dissection as described in the written portion of this protocol. The finished pipette should have a smooth, shallow taper and a 45 degree bevel with an internal diameter of approximately 100 micrometers.

Backfill the pipette of mineral oil by capillary action about halfway. Insert a vacuum grease dipped plunger into the back of the pipette. Then push it in to advance the meniscus of the oil to the pipette tip.

Secure the pipette with plunger to a micro manipulator on an adjustable arm. Front load the pipette with a solution of 50%fluorescent microbeads, 5%glycerol and 45%water. Freshly dissect a hole mount as shown previously, and transfer it ventricle side up to a clean dissection dish filled with 37 degrees Celsius.

Leitz medium, immobilize the hole mount to the dissecting dish using two insect pins in the thalamus and the anterior dorsal cortex. Place the base of the stationary arm holding the micro manipulator next to the stereo microscope, keeping the arm elevated to avoid breaking the micro pipette needle. Dip the tip of the micro pipette into a tube of medium to clean microbeads off the exterior of the tip.

Being careful not to break the tip. Position the tip of the pipettes over the dorsal surface of the lateral wall and lower the arm to bring the tip of the pipette just above the tissue surface. Focus on a field of view encompassing the anterior half of the hole mount, and begin recording images of desired.

Use the plunger to eject approximately five liters of the micro beads onto the hole mount surface. Once the initial bolus of beads has been cleared off the surface by a penal flow, use the plunger to eject additional five nanoliter rounds of microbeads. Flow, direction and speed can be analyzed using the recorded video.

The final section of this video demonstrates how hole mounts are immuno stained and mounted onto slides for confocal imaging. Freshly dissected hole mounts are first transferred to a 24 well plate and fixed by immersion in paraform aldehyde. This is followed by a series of incubations in primary and then fluorescently conjugated secondary antibodies with intervening washes.

After staining, the whole mounts are sub dissected to separate only the thin layer of the lateral wall containing the subventricular zone away from the remainder of the hole mount containing the underlying st stratum and cortex. This thin flat piece of tissue is then transferred to a slide and cover slipped to fix hole mounts for immuno staining chill. A 24 well plate containing 4%paraldehyde and 0.1%tritton X 100 on ice.

In a chemical fume hood, gently immers a dissected hole mount ventricle side up within one well of the plate. Fix the sample overnight at four degrees Celsius, aspirate the fixative and use a transfer pipet to carefully wash the hole mount in one XPBS with 0.1%Triton X 103 times for five minutes each. For all liquid handling steps, pipette gently at the side of the well to avoid flipping the hole mount and damaging the tissue.

Add a blocking solution containing triton X and 10%Normal goat serum in one XPBS use up to 2%Triton X.Depending on the required depth of antibody penetration. Incubate the whole mount in blocking solution for one hour. At room temperature, remove the blocking solution.

Add primary antibodies diluted in blocking solution and incubate a four degrees Celsius for 24 or 48 hours depending on the target antigen wash with two quick rinses of one XPBS with triton X, followed by three washes of 20 minutes each at room temperature, add secondary antibodies diluted in blocking solution and incubate a four degrees Celsius for the same amount of time as for the primary antibody wash in the same manner as for the primary antibodies. The next series of steps demonstrate how to dissect only the lateral wall from the remainder of the whole mount as a single flat specimen. That can then be mounted for high resolution imaging.

Return the immunostain hole mount ventricle site up to a clean dissecting dish filled with one XPBS under the stereo microscope and moving from posterior to anterior. Use a stab knife to remove the dorsal cortex, an underlying white corpus callosum in order to visualize the interface between the sub ventricular zone and the stri aum. Then make a long horizontal cut across the ventral aspect of the whole mount.

Use fine forceps to orient the tissue on this new flat edge so that the dorsal surface is facing up. The subventricular zone is a thin band of homogeneously pink tissue while the striatum is infiltrated by cords of white matter. Use the forceps as two pins in the stri AUM to anchor the tissue without damaging the ventricular wall.

Beginning anteriorly. Use the stab knife to carefully begin cutting between the striatum and the subventricular zone. It is important to keep the cuts parallel to the ventricular surface and to maintain the thickness of the dissected tissue.

As the sub ventricular zone thins posteriorly. Carefully remove all surrounding tissues from the hole mounts that are not part of the ventricular wall. The hole mount should be of a uniform thickness between 200 and 300 micrometers.

Use forceps to lift up the sliver of tissue from below and transfer it to a microscope Slide. Apply mounting medium, then a cover slip. Try not to introduce air bubbles.

Store the slides flat in a slide book at four degrees Celsius for one to two days to allow the cover slips to settle before imaging The samples. Here are some representative results for whole Mount Immunos staining and append flow assays. The first image is a low power view of a whole mount stain for double cottin, a marker of neuroblast that demonstrates the network of chains of migrating young neurons in the adult subventricular zone.

Next is another low power view of a hole mount that was stained for KI 67 in green and blood vessels in red illustrating the recently described close interaction between dividing cells and the vasculature in the subventricular zone at higher power. The next picture shows a confocal image of the ventricular surface with the apical surface of cells lining the ventricle outlined by beta catine in staining in green and the basal bodies of those cells labeled by gamma tubulin in red. The final high power image is a maximum projection of a confocal stack taken from a whole mount stain for GFAP showing the long GFAP positive basal fiber of S vz type B one cells to analyze CSF flow, direction, and speed on the ventricular surface.

Consecutive frames from append flow movies can be merged into a single composite image. These composite images show the position of single beads at different points in time as they traverse the append or surface. Here is an example of how the results look when complete.

You have just seen how to prepare whole mounts from the subventricular zone of the lateral wall of the lateral ventricle. This provides whole mounts view of the entire wall and has allowed many discoveries over the years, including the observation that this ventricular so is organized as a network of pathways for chain migration. It also allowed us to study how this service panel fluid flows over this wall, and indirectly also suggested ways in which these cells guide their migration through these very extensive network of pathways.

More recently, this technique has allowed us to discover that the sub ventricular so is organized as pin wheels with the stem cells in the core of these pin wheels revealing the optical ending of the stem cells within this structure. Since the whole mount allows a whole perspective of this ventricular zone, it provides a a view that sections do not provide, and for this reason, we're making it available for everyone as it should be useful for many other studies. There are a few keys to remember when preparing hole mounts that will improve your dissections.

The first is to have proper lighting adjust your lighting to cast shadows on the hole mount. That'll provide contrast and show you where you should make your cuts. The second is to use the forceps as two mobile pins.

You don't wanna use the forceps to squeeze the tissue or lift up the tissue. Rather use them as two mobile pins that'll secure the tissue while you're cutting. The third is to use a combination of general retraction and cutting.

As you notice in the dissections, very little cutting of tissue is actually done. Most of the dissection is done by general retraction during the second round of the dissection. When you're taking off the lateral wall from the remainder of the whole mount.

The most important thing is to orient your cuts in a plane that's parallel to the plane of the ventricular surface. That'll ensure that you have a single thickness from anterior to posterior along the lateral wall. And that will also ensure that when you're doing confocal high resolution imaging, you'll have images that all come into focus in a single plane.

We hope this video will be useful for you for your studies. Good luck with your experiments.