I am Troy Gasai. We're gonna talk about an organotypic slice assay that's been perfected in my lab over the last few years. Ben jacket in my lab will demonstrate to you how this assay is performed.
The organotypic slice assay of the postnatal brain is a powerful method for testing various factors and the role on neuronal migration in the postnatal brain and the TRO migratory stream. Hi, I'm Ben Jakay. I'm a postdoctoral fellow in Dr.Troy gge lab at the College of Veterinary Medicine of North Carolina State University, and I'm here today to show you how we perform tissue cross transplantation between organotypic mouse brain slices in order to visualize migrating neuroblast in the postnatal brain.
All techniques described in this video should be performed in a sterile environment, preferably a laminar flow hood using sterilized tools. A 150 microliter drop of slice medium is placed in the center of the glass, bottom part of the dish with a disposable syringe equipped with a 23 gauge needle. Several glue spots are placed outside the circular glass bottom cover slip while leaving one side unglued for fluid exchange A.A nuclear pore membrane is then gently placed on top of the medium using micro forceps while ensuring that air bubbles are not trapped underneath the membrane.
The curved forceps are then used to flatten the glue spots and secure the membrane in place. One milliliter of slice medium is added on top of the membrane, and finally, the dishes are placed in an incubator until ready to use. The best results are obtained when slices are prepared from young postnatal mice, P one through P ten first, the head is stabilized by holding the snout with micro forceps.
The skin is excised longitudinally from the neck to the snout. The scalp is peeled away to expose the bone and the skull is cut longitudinally and anteriorly starting at the sternum magna by making one medial and two lateral cuts, one on each side. Care should be taken to minimize contact with underlying cortical tissue.
As the cranial flaps are removed away from the brain to improve stability of the tissue. During the vibram sectioning, the lateral most and codal aspects of the brain are removed with a razor blade. The two hemispheres are carefully scooped out of the skull and placed medial surface down in an embedding mold.
They are immediately covered with melted 3%DNA grade AGA roses gel dissolved in tissue preparation buffer that is maintained at 37 degrees Celsius. After two minutes of stabilization on a flat horizontal surface. To ensure even hardening of the aga roses, the molds are positioned on ice to complete the setting.
Once the gel containing the hemispheres is set, it is removed from the mold and trimmed around the brain tissue. The gel embedded tissue is then mounted on the specimen disc of the vibrate tone with a medial surface up and secured with cyanoacrylate adhesive. The disc is then installed in the vibration specimen tray filled with ice cold tissue preparation medium, and the brain is sectioned at 150 micrometer thickness.
Once RMS containing slices are released from the blade, they're carefully scooped out using a small flathead spatula and placed flat at the center of the culture dishes. It is critical that handling the slices is kept to a minimum as the tissue is very fragile. The amount of medium in the culture dishes is adjusted to the level sufficient to cover the brain sections while preventing them from moving around.
The dishes are labeled and transferred to an incubator. Donor brains are prepared in a fashion similar to that of recipient brains, except that the sections are cut at 250. Micrometer thickness and slices are collected in ice.
Cold tissue preparation. Buffer slices are immediately placed under a dissecting microscope with epi fluorescence capability. The RMS is visible as a gray U-shaped structure extending from the subependymal zone to the olfactory bulb.
The RMS is gently microdisect using micro forceps. One forceps is used to stabilize the slice while the other is used to make small cuts around the RMS until it is released from the slice. The excised RMS is then cut into small x explan of approximately 200 to 500 micrometers in diameter.
In this example, eggplants are prepared from mice expressing the red fluorescent protein, TD tomato glass. Bottom dishes containing host slices are removed from the incubator and placed under the dissecting microscope using visible light. A small incision is made in the initial segment of the RMS and using a pipeter equipped with a 20 microliter tip, A single donor RMS Explan is transferred to the incised site in the host RMS.
The explan is gently pushed into the incision to establish contact between the two tissues. To ensure this contact is stable, the explan are pushed slightly in between the slice and the nuclear poor membrane. The dishes are then returned to the incubator for at least one hour to allow the sections to settle on the membrane.
Neuroblast should begin to migrate from the explan into the host RMS after approximately one to two hours. Cell migration is best visualized by using extra long working distance 20 power objectives. The tissue cultured issues are transferred from the incubator to an incubated chamber on the microscope.
Fluorescent neuroblast can be imaged at intervals ranging from zero to 10 minutes depending on the type of analysis desired. Our organic typic slice culture protocol has been thoroughly tested and optimized over the last few years for consistency in migration pattern and orientation In the RMS. In this example, analysis of cells immigrating from explan, obtained from mice in which TD tomato is expressed under the nest in promoter reveals highly oriented and rapid migration into the host RMS.
High magnification. Time lapse analysis shows excellent resolution of the entire length of a migrating neuroblast during a 20 minute imaging session. Once the imaging is completed, slices skin be fixed with ice, cold and freshly prepared, 4%paraform, aldehyde, and immunostain for different components of the migration stream.
In this case, GFAP positive astrocytes in blue and CD 31 positive blood vessels in green are revealed using fluorescent immunohistochemistry, high magnification analysis of slices stained for the cytoskeletal proteins actin in blue, and tubulin in green reveal non-uniform expression of these components by a cell. In the midst of migration. Our protocol presents some challenges that require patience, practice and committed time to both the preparation and the analysis of your results.
So here are some useful suggestions that will help you get the best results out of your experiments. In our experience, mice in the age range of P one to P 10 provide the best results. Age matching between donor and recipient brains also improves the reproducibility of the experiments.
Since most reagents and culture dishes must be freshly prepared, our slice assay requires several hours of uninterrupted preparation and imaging in a single day. Once the brains are harvested, consequence steps must be accomplished as fast as possible. Between decapitation and preparation of slices, all steps should be performed using ice, cold reagents and tissue manipulation should be kept to a minimum in order to avoid anatomical disruptions.
The tools utilized for these dissections should be sterile and reserved for live tissue manipulations only. Never utilize tools that were ever in contact with fixatives such as formaldehyde. The slices are generally viable for up to 36 hours with visible decline in migration, speed, and orientation between 24 and 36 hours.
Beware of this limitation when planning your experiments and analysis.
