The overall goal of this procedure is to demonstrate how to obtain enriched populations of hippocampal neurons and single neurons from frozen tissue sections by laser capture microdissection for subsequent gene expression analysis. This is accomplished by first anesthetizing, a rat in an anesthetic chamber. The rat is then intubated, mechanically ventilated and placed in a stereotaxic head holder to prepare the rat For fluid percussion brain injury, a craniotomy is performed.
The second step is to administer the fluid percussion brain injury. Next coronal sections of the brain are cut on a cryostat and stained with either fluro aid, a marker for degenerating neurons, orreal violet, a missile stain. The final step is to perform laser capture micro dissection and collect single neurons or pools of neurons.
Ultimately, total RNA is isolated from the captured cells, analyzed for quality and quantified using an Agilent bioanalyzer and used in downstream applications such as quantitative real-time PCR and pathway focused or genome-wide microarray analysis. The main advantage of this technique of existing methods like manual micro dissection, is that we can obtain enriched populations of neurons or other brain cell types such as glia or immunomodulatory cells, or single neurons from tissue sections. This allows us to perform gene expression analysis in distinct identified cell types in the brain and in other heterogeneous tissues composed of multiple cell types, Demonstrating this procedure will be missed.
Debbie Boone, a senior research associate in our laboratory. First retrieve the brain tissue obtained from rats with fluid percussion, traumatic brain injury, or sham injury from the minus 80 degrees Celsius freezer, and transfers to the minus 22 degrees Celsius. Cryostat for 10 minutes to bring the brain to sectioning temperature while the brain is thawing.
Wipe the cryostat with RNA zap containing ethanol and clean the brushes with ethanol. After the 10 minutes of elapsed, remove the brain from the tube and place it ventral side up onto a piece of gauze on the cryostat stage, use a fresh razor blade and slice the brain to remove the posterior portion of the brain. Just rostral to the cerebellum and the anterior portion at the optic chiasm.
Fill a cryo mold with OCT mounting medium and place the brain into the mold with the anterior side down. Allow the brain tissue to freeze in the mounting medium until it turns white. This should take approximately 10 minutes.
Now freeze the specimen disc onto the brain with OCT. Then remove the mold. Insert the disc with the brain attached into the specimen head and tighten the screws.
Insert a disposable low profile blade into the knife holder and tighten the lever down. Begin slicing the brain at 20 microns to remove the outer layer of OCT. Once the hippocampal region is reached, set the micron dial to 10.
Collect coronal serial sections by placing a glass slide or plus glass. Slide onto the tissue section. Place the slides in an RNAs free staining rack at minus 20 degrees Celsius until sectioning is complete.
Before proceeding to stain the sections, wipe all staining containers and graduated cylinders with ese and rinse in milli Q water. Ensure that all solutions have been prepared with RNAs free water and that the crestal violet and fluoro jade stain have passed through a 0.2 micron filter before. Use Thor brain sections at room temperature for 30 seconds and fix in 75%ethanol for one minute.
Then proceed to the appropriate counter stain for LCM of either swaths of neurons or individual neurons for LCM of swaths of neurons after fixation, rinse slides in RNAs free water for one minute stain with 1%crest violet for one minute. Rinse in RNAs free water three times for one minute each dehydrate with 95%ethanol twice for 30 seconds each. Then 100%ethanol twice for 30 seconds and xylene twice for three minutes each.
After staining air dry all sections in a fume hood for 15 minutes and then proceed to LCM.Here. LCM is performed. Using a P cell two E microscope with an infrared diode laser first adjust the center joystick to the vertical position.
Then rotate and lock the Forex objective into position. Place a slide with stained hippocampal sections at the center of the microscope stage and manually adjust until the region of the hippocampus is in view. Use course and find focus adjustments.
To bring the section into focus, activate the vacuum chuck by pressing the vacuum button on the controller. Then rotate and lock the 10 x objective into place. Focus again with course and fine adjustments.
Load the caps your macro LCM caps into the caps, your cassette module, and position a cap at the load line position. Rotate the cap placement arm and position around the cap. Then raise the cap placement arm to remove the caps your cap from the cassette module.
Rotate the cap placement arm over the slide and then lower it down over the slide to place the cap onto the slide. Adjust the fine focus and move the joystick to check if cells are inside the black circle on the cap. All cells to be captured should be inside this black circle.
Now set the laser parameters. First press the laser enable button on the controller. The laser target spot will be visible as a pink.
in the field of view on the computer monitor. Set the laser spot size to small to focus the laser and adjust the power and duration to 65 to 75 milliwatts for one to two milliseconds as needed for optimal cell capture. To test the laser, use the joystick to move the laser spot to an area where there are no cells to pick up.
Fire the laser using the thumb switch. Then use the joystick to move the laser spot away from the melted polymer spot and check to see if there is a visible dark ring surrounding a clear area where the laser was fired. To capture cells, use the joystick to move the laser spot over the area of cells to capture and fire the laser.
Using the thumb switch, move the joystick while firing the laser to capture a large area of cells. After firing all cells of interest, lift the cap placement arm. The captured cells will separate from the tissue section and adhere to the cap.
Your cap, the remaining tissue and missing cells will be visible in the field of view. The cells on the cap can also be viewed by placing the cap on an empty portion of the slide. Lift the cap placement arm and rotate it to the cap unload station.
Lower the cap into the station and rotate the cap placement arm back into its previous position. Slide the cap your insertion tool along the platform and onto the cap. Lift the tool with the cap attached from the platform.
Lightly touch the cap to the adhesive part of a post-it note to clean away unwanted tissue. Place the cap into a 0.5 milliliter RNAs free tube filled with 100 microliters of lysis buffer obtained from the RN aqueous micro isolation kit. Immediately vortex the cap for 30 seconds to lies.
The cells incubate the samples at 42 degrees Celsius for 30 minutes and freeze at minus 80 degrees Celsius until RNA isolation. The staining procedure for LCM of single injured neurons after fixation is similar to the procedure for swaths of neurons. However, some incubation times differ and the sections sustained with fluoride for four minutes before rinsing and dehydration, see the text portion of the protocol for details after staining.
Again, air dry all sections in a fume hood for 15 minutes. Before proceeding to LCM to capture single cells load capture HS LCM caps into the cap or cassette module using the same procedure as for the macro LCM cap. Again, set the laser spot size to small to focus the laser, but this time adjust the laser power and duration to 65 to 75 milliwatts for 0.45 to 0.5 milliseconds for optimal cell capture of single cells.
Rotate the cap placement arm over the slide and lower the arm down towards the slide. To place the cap over the slide, the capture HS cap surface sits elevated from the sample. During LCM, use the joystick to move the laser over single Fluor aid positive cells.
All cells being captured must be inside the small black ring on the cap fire the laser using the thumb switch. When all cells within the black circle area have been fired with the laser lift, the cap placement arm, the captured cells will separate from the tissue section and adhere to the capture hs cap. Now place another slide on the microscope stage and collect fluro aid positive cells until the HS cap is full.
Then place the cap into a 0.5 milliliter RNAs free tube filled with 40 to 50 microliters of lysis, buffer, and vortex and incubators before again freeze at minus 80 degrees Celsius. If not immediately performing RNA isolation according to the instructions in the text portion of the protocol. The following images show laser capture microdissection of swaths of specific cell populations from the rat brain frozen 10 micron coronal sections of the rat brain.
Were prepared according to this protocol and stained with crestal violet and missile stain. This image shows parametal neurons from the ca one to ca three subfields of the rat hippocampus. Here, granule cells in the hippocampal dentate gyrus have been captured.
Finally, we show swaths of neurons captured from the bilateral supra chiasma nuclei located on either side of the third ventricle and above the optic chiasma. The SCN contains the master circadian clock that controls circadian rhythms throughout the body. The following images show laser capture microdissection of single neurons from rat hippocampus.
Shown here are degenerating fluoride positive hippocampal ca three neurons here surviving fluro aid negative ca three neurons before and after LCM are shown captured cells are visualized on the LCM cap. The following images showed gene expression analysis of total RNA, isolated from laser captured neurons. This image shows quantitative real-time PCR data of circadian clock gene expression in the SCN.
This heat map of gene expression in dying and surviving hippocampal neurons shows expression profiling of apoptosis related genes using focused PCR arrays. Finally, we show an example of a heat map generated from a whole genome ENT microarray analysis of gene expression in ca one to ca three neurons from rats that received sham injury TBI or TBI plus a recumbent adeno-associated siRNA virus designed to knock down expression of either neuronal nitric oxide, syntase, or glutathione peroxidase one, both of which are genes induced by brain injury After its development. These techniques paved the way for scientists in many fields of research, including brain injury and cancer who use animal models of these disorders to perform genomic proteomic and metabolic profiling of specific cell populations.
For example, we are currently using these techniques to laser capture specific cell populations from the prefrontal cortex and nucleus accumbens brain areas that are known to be involved in addiction.
