The overall goal of this procedure is to demonstrate how to slice and dissect the supra matic nucleus from transgenic mice and to culture the schematic tissue in order to record clock gene or protein expression as luciferase induced by luminescence. This is accomplished by first dissecting out the brain from a transgenic mouse that contains a luciferase reporter for a clock gene or protein, and then making a brain slice containing the supra matic nucleus. The second step of the procedure is to dissect out the small bilateral schematic nuclei from the slice.
The third step of the procedure is to culture the schematic nucleus explan on a membrane in a Petri dish that contains culture, medium and luciferian. The final step of the procedure is to record luciferase activity by measuring bioluminescence with photomultiplier tubes in a light tight heated chamber. Ultimately, results can be obtained that chose circadian oscillation of the clock gene or protein.
My name is Sergey IV and I'm working in Gabrielle Re Club. My name is an SoFi Johansson and I'm working in Gabriella's team. My name is Gabriel Quist.
I am a principal investigator at the Department of Neuroscience, And my name is Ka LaShon, and I'm a PhD student at the Swedish Medical Neuroscience Center, and I Collaborate with Gabriella. The main advantage of this technique over existing methods like Western Blot R-T-P-C-R, and in two hybridization is that expression of a clock gene or protein can be followed continuously for many days in the same tissue with very high resolution of about one minute, allowing detailed analysis of period amplitude, and face of the circadian Rhythm. Visual demonstration of this method is critical as the SEN slicing and dissection steps are difficult to learn.
The SEN is very small and can be difficult to identify. Demonstrating the procedure will be Dr.Sge Velia, a postdoctoral fellow in my research team. After making the necessary solutions, prepare the Petri dishes that will be used in the experiment using a five milliliter syringe prefilled with autoclave vacuum grease.
Apply the grease on the top ring surface of a 35 millimeter Petri dish. Prepare a separate Petri dish for each slice.Culture. Fill a Falcon tube with 10 milliliters of air, buffered culture medium.
This will make enough medium for eight cultures. Add 10 microliters of freshly thawed luciferian. The luciferian is light sensitive, so protect the luciferian medium from light by storing the Falcon tube in a dark 37 degree Celsius heating chamber.
Next, sterilize all instruments, cover glasses and other material to be used in the procedure. Spray all non-sterile equipment with 70%ethanol. Expose the instruments and the greased petri dishes to UV in a sterile hood for at least 30 minutes.
Another preparatory step is to set up the vibrato and attach a sterile razor blade. The equipment is now set up for the surgical procedure. After anesthetizing and decapitating a mouse, remove the eyes from the head with a pair of scissors.
This prevents training or exciting the optic nerves, which helps avoid damage to the schematic nucleus or SCN. Use a fine pair of scissors to remove the last cervical vertebra and to remove the skin. Make two cuts in the skull bone along both sides of the skull, thus making a removable lid.
Open the skull with a micro Ron Jre tool. Use the RJ with an upward motion never pressing down on the brain with the tool. In order to prevent damage to the coddle SCN, remove all bone until the olfactory bulbs can be seen.
Use a fine micro dissecting spring scissors to carefully cut through the optic nerve between the hemispheres and the olfactory bulbs.Please. A Petri dish filled with 50 to 100 milliliters of cold HBSS under the mouse's head. Turn the head upside down and let the intact brain start to fall out.
Cut other cranial nerves as necessary on the opposite side from the optic nerve. When detached, the brain falls into the Petri dish, which rapidly cools the brain. Use a spoon to transfer the ice cold brain to a sterile cutting surface, such as a glass Petri dish lid turned upside down.
Remove the cerebellum by making a perpendicular cut with sterile razor blades between the hemispheres and the cerebellum. Apply superglue on the dry platform of the viome. Insert a sharp curved forceps in the rostral part of the brain and pick up the brain holding the brain with the forceps.
Carefully dry up the HBSS on sterile filter paper. Fix the hemispheres onto the glue on the platform with the rostral tip upwards and ventral surface closest to the cutting blade. Attach the platform in the vibrato holder and immediately fill it up with cold HBSS.
If the perpendicular cut is made properly, the hemispheres should stand straight up. The goal is to cut coronal slices containing the mid region of the bilateral SCN, using the maximum speed of the vibrato. Start cutting off 500 to 800 micrometer thick sections of the hemispheres until reaching the commissure.Watch.
As the optic chiasm gets wider, accompanied by reduction of sections to 100 micrometer thickness, continue slicing down in the coddle direction, slowing down the horizontal manual movement of the blade until the two s SCN nuclei start to appear. Use a magnifying glass if necessary to visualize the nuclei when the level of the SCN has been reached. Cut an SCN section of 250 micrometers.
Fill a lid from a medium sized Petri dish with cold HBSS using a soft brush. Lift and transfer the SCN brain section to the Petri dish. Place the Petri dish under a dissecting microscope.
The next step is to dissect the SCN out of the slice. Use the microscope to verify that the bilateral SCN is clearly visible on at least one side of the section. Use a pair of sterile scalpels to dissect out a square of tissue, about 1.5 millimeters on each side containing both SCNs.
Cut as close to the SCN as possible. Without removing SCN tissue, a small piece of the optic nerve must be attached to the explan, but no other nuclei should be included. Cut the tissue containing the bilateral SCN in half to obtain two unilateral SCNs.
One unilateral SCN can be used as a control in a sterile hood. With the light off, add 1200 microliters of the Lucifer medium to a 35 millimeter diameter Petri dish, and then place a culture membrane on top of the liquid surface. Make sure there are no air bubbles under the membrane.
The implants are tiny and difficult to pick up. Therefore, use a 1000 microliter pipette with tip to suck the SEN explan into the tip and press it out on the culture membrane. Discard excess HBSS for luciferase recording place only one SEN per Petri dish.
Seal the grease Petri dish with a 40 millimeter diameter cover glass. Add more grease if the seal is not tight. Transfer the Petri dishes to aluma cycle apparatus placed in a 36 to 37 degrees Celsius light tight heating chamber.
To begin culturing, note the culture dishes and photomultiplier tube detector assemblies mounted inside the lum cycle chamber. Place the culture dishes below the photomultiplier tubes approximately one to two centimeters. The photomultiplier tubes detect and amplify the luciferase induced by luminescence.
The Luma Cycle software tool is used for data acquisition. Photon counts are integrated over one to 10 minute intervals to get high resolution of the gene protein expression. The circadian expression of the gene protein is analyzed to determine phase period and amplitude of the rhythm.
This figure shows bioluminescence recordings from healthy and non-healthy SCN tissue cultures. These mice were held in 12 hours light, 12 hours dark prior to the removal of their SCN tissue. The black line shows the circadian rhythm of the bioluminescence recordings of period two luciferase expression.
In a healthy SEN slice, the protein oscillates with the circadian variation in which the maximum expression of the protein occurs at ze gaber time, 12 to 13 or the onset of darkness. Thus, the phase of the gene rhythm is dependent on the light dark schedule in which the animal was kept before sacrifice. The blue line shows bioluminescence recordings from an SE and culture that dried out after the culture dish was opened at day four indicated by the arrow and not resealed properly.
The red line shows bioluminescence recordings from a non-healthy SCN culture. This figure uses the SCN culture in a pharmacological experiment. The figure shows a trace from a culture that was treated with an HCN ion channel blocker at the time indicated by the first arrow.
Over the course of two to three days, the ion channel blocker reduced and then stopped the circadian oscillation at the second arrow. The drug was washed out and replaced with conditioned control medium While attempting this procedure. It's important to remember to work quickly but carefully in order to keep the SEN tissue viable.
It's also important to standardize your vibram sectioning so that the same level of the SEN is obtained each time. After watching this video, you should have a good understanding of how to slice and dissect the SEN culture, the SCN organ, typically on a membrane and monitor clock gene or protein expression as luciferase induced by luminescence in the cultures.
