This technique makes it possible to give a closer look to single cells in living tissue. By tracking and manipulating single cells, we can better understand tissue formation during development. Visual demonstration of this method enables potential users to decide if this technique could be helpful for their work.
Additionally, it makes it possible for users to see how the application is run, which would be difficult to grasp from the text alone. This protocol can be applied to every tissue with an assessable surface. We have been targeting different cells in the same tissues, different tissue in the same species, and also different species.
When attempting this protocol, it is important to remain focused and work quickly. All equipment should be prepared before starting the dissection. Begin by installing the auto-injector software and pulling the microinjection pipettes from borosilicate glass capillaries with the micropipette puller.
Store the pipettes for up to three days protected from dust. Melt 3%wide range agarose using a microwave oven. Do not let the agarose solidify by keeping it in a water bath at 37 degrees Celsius.
Thaw an aliquot a SCM and warm 10 to 12 milliliters SIM and 20 milliliters of Tyrode's solution to 37 degrees Celsius using a water bath. Mix the fluorescent tracer with the other chemicals to be injected, then centrifuge the microinjection solution at 16, 000 times G for 30 minutes at four degrees Celsius. Collect the supernatant and transfer it into a new tube.
Keep the microinjection solution on ice until use. Use the heads from E13.5 to E16.5 mouse embryos to prepare organotypic tissue slices of the telencephalon. Remove the skin and open the skull with forceps moving along the midline.
Dissect out the embryonic brain from the open skull and remove the meninges covering the brain tissue starting from the ventral side of the brain. Leave the dissected whole brain in Tyrode's solution on the 37 degrees Celsius heating block. Pour the wide range melted agarose into a disposable embedding mold.
When it cools to 38 to 39 degrees Celsius, carefully transfer up to four brains into the agarose using a Pasteur pipette. Stir the agarose around the tissue with a spatula or a pair of Dumont number one forceps without touching the tissue. Let the agarose solidify at room temperature.
Once the agarose has solidified, trim the excess surrounding the tissue. Fill the buffer tray with PBS. Orient the brain with the rostral caudal axis of the tissue perpendicular to the tray and cut 250 micrometer slices using a vibratome.
Fill a 3.5 centimeter Petri dish with two milliliters of pre-warmed media, then use a plastic Pasteur pipette to transfer 10 to 15 slices to this dish. When finished, transfer the Petri dish with the slices into the slice culture incubator. Maintain the slices at 37 degrees Celsius in a humidified atmosphere.
Turn on the computer, microscope, microscope camera, manipulators, pressure rig, and pressure sensor. Load the application by clicking the file, launch app. py in the main folder downloaded from GitHub and specify the device settings in the popup screen.
To prevent unwanted clogging, create an outward pressure before submerging the pipette into the solution. Slide the compensation pressure bar to 24 to 45%and click set values. Next, tune the pressure by turning the mechanical pressure valve knob to one to two PSI as indicated by the pressure sensor.
Transfer the slices to the center of a 3.5 centimeter Petri dish containing two milliliters of pre-warmed SIM, then place the Petri dish on the microinjection stage that has been preheated to 37 degrees Celsius. Load the microinjection pipette with 1.4 to 1.6 microliters of microinjection solution using a long tip plastic pipette and insert the microinjection pipette into the pipette holder. Using the lowest magnification on the microscope, bring the slice into focus and guide the micropipette to this field of view so that it is focused on the same plane as the slice target.
Switch the output of the microscope to the camera to see the FOV and the application. Click the magnification button in the top left of the interface to initiate device calibration. When a window prompts to select the magnification, select the 10X and press OK.The software assumes the internal objective lens is 10X.
Refocus the pipette tip using the micrometric wheel of the microscope and click the pipette tip with the cursor. Next, press the step 1.1 button and press OK in the popup window. The pipette will move in the y-direction.
Click the tip of the pipette and press the step 1.2 button. Lastly, enter 45 in the pipette angle box and press the set angle. Enter desired parameters into the automated microinjection controls panel and set the speed to 100%When finished, click set values.
Click the draw edge button and drag the cursor along the desired trajectory in the popup window to define the trajectory of injection. For microinjecting neurons, target the basal side of the telencephalon. Bring the pipette to the start of the line and click its tip, then click run trajectory to start microinjecting.
Repeat this step for every plane of injection targeted. Immerse the slices into the collagen mixture, then transfer the slices together with 200 to 300 microliters of the collagen into a 14 millimeter well of a 35 millimeter glass bottom dish. Ensure that the slices are covered in the least amount of collagen possible, which is the optimal condition for nutrients and oxygen uptake.
Orient the slices using two pairs of forceps and incubate the Petri dish for five minutes at 37 degrees Celsius on a heating block to allow the collagen to solidify. Move the Petri dish back to the slice culture incubator for an additional 40 minutes, then add two milliliters of pre-warmed SCM. At the end of the culture, take the slices out of the slice culture incubator and aspirate the SCM.
Wash the collagen-embedded slices with PBS, then add 4%paraformaldehyde and leave the tissue at room temperature for 30 minutes. Move it to four degrees Celsius for overnight fixation. On the next day, aspirate the paraformaldehyde solution and wash the slices with PBS.
Remove the slices from the collagen using two pairs of forceps under a stereo microscope. Use a microwave to melt the 3%low melting point agarose, then pour it into a disposable embedding mold and let it cool to approximately 38 or 39 degrees Celsius. Transfer the tissue slices into this mold making sure that the peel side of the slice is facing up, then allow the agarose to cool to room temperature and solidify.
Trim the extra agarose surrounding the slices. Orient the agarose block to ensure that the cut surface is parallel to the vibratome's cutting blade and cut 50 micrometer thick sections. Fill a 24-well dish with PBS and transfer the sections into this dish using a fine-tip paintbrush, then perform immunofluorescence according to standard protocols.
Representative images of successfully injected progenitor cells and newborn neurons are shown here. When injected with Dextran Alexa 488, cells appear fully filled with the dye. Automated microinjection can provide significantly higher throughput compared to manual microinjection.
Furthermore, EDU labeling confirms that cell viability is not affected by automation. Keeping the organotypic slice in culture makes it possible to follow lineage progression of the microinjected cells. Microinjection into single neural stem cells provides excellent single cell resolution.
Therefore, it has been used to dissect the cell biology of neural stem cell progression and fate transition. This protocol was used to study junctional coupling in newborn neurons by injecting Lucifer yellow along with Dextran A555. A proportion of newborn pyramidal neurons were coupled via gap junctions to neighboring neurons, which is consistent with the idea that immature neurons communicate via gap junction.
We have used this technique to investigate the cell biology of brain development and brain evolution. We studied several candidate genes that were affecting neural stem cell behavior and we were able to discover and understand how they work, how they the affect neural stem cell lineage progression, and the neuron production during brain development.
