My name is Gail Siebel and I'm a postdoctoral fellow in the laboratory of Dr.Veronica Alvarez at the National Institute on Alcohol Abuse and Alcoholism. This work was performed in collaboration with Dr.Kathleen Grant and Andrew Wow at the Oregon Primate Center and Dr.James Dene at Wake Forest University, who provided the non-human primate tissue used in this study. The overhaul goal of this procedure is to introduce fluorescent dyes into neurons and label brain sections from different species such as rodents and primates of any age.
For the purpose of studying the morphology of dendrites and dendritic spines, this is accomplished by first coating Ts and beads with a lipophilic dye such as D eye. The second step of the procedure is to line tubing with D eye coated beads and cut it into small pieces which serve as bullets for the Helios gene gun system. The third step of the procedure is to shoot D eye coated beads into brain slices using the gene gun.
The final step of the procedure is optional as stylistics can be combined with immuno staining to simultaneously localize other markers. Ultimately, results can be obtained that show sparse fluorescent labeling of neurons suitable for high resolution imaging, such as confocal or two photon microscopy, and the study of dendritic branching and dendritic spine morphology. The main advantage of this technique over our existing methods is that stylistics can be applied to brain sections from animals of all species, ages and genotypes.
It can be used in combination with immunostaining and produces sparse fluorescent labeling that's suitable for high resolution microscopy Before making bullets. Coat tungsten beads with lipophilic dye in a chemical fume hood at 450 milliliters of methylene chloride to 13.5 milligrams of lipophilic dye dye eye for a final concentration of 30 milligrams per milliliter. Take one pre Eloqua tube containing 300 milligrams of tungsten beads and add 300 microliters of methylene chloride pipe it vigorously to resuspend the beads and make a homogeneous solution.
Only 100 milligrams of tungsten beads are used per bullet set pipetting up and down to keep the beads resuspended, add 100 milliliters of the beads and the dissolved die die eye onto a glass slide and mix them together thoroughly. With a pipette tip, allow to air dry completely until the beads turn light gray. Place a clean piece of waxed whitepaper under the slide using a clean racer blade For each color of bullets, scrape the beads off the glass, slide and D dice them into a fine powder.
Scrape coated beads onto whey paper and funnel them into a 15 milliliter conical tube. Add three milliliters of water and sonicate in the water bath for 10 to 30 minutes at room temperature to thoroughly disrupt any clumps. Cut 0.7 meters of teal tubing with an angle on one end and connect the other end to a 12 milliliter syringe using a tubing adapter, place the free end into a tube containing 10 milliliters of a 10 milligram per milliliter polyvinyl perone or PVP solution, and draw the solution completely through the tubing and into the syringe.
Vortex the bead solution while pumping with the syringe to make a homogeneous mixture working quickly to avoid precipitation of the beads, draw the solution completely into the tubing. Avoid introducing any air bubbles, which will prevent the beads from attaching to the tubing in that spot while holding the tubing taut from both ends. Tilt it from side to side to evenly.
Distribute the beads. Feed the tubing into the prep station and allow the beads to settle for 30 to 60 seconds. Use the syringe to slowly but smoothly.
Remove the water leaving the beads behind. Immediately disconnect the syringe and begin spinning the tubing. Adjust the nitrogen flow on the prep station to four to five LPM dry for 10 to 20 minutes until water droplets are no longer visible.
Remove the tubing from the prep station, cut bullets into 13 millimeter lengths with the tubing cutter observed and on good bullets will have a faint gray haze uniformly coating the interior. Place the bullets in foil wrapped scintillation vials containing a drying agent such as anhydrous calcium sulfate. Store it room temperature until ready to shoot.
Diic Labeling can be used to stain neurons in brain tissue from diverse species and ages and preserved using various methods. Place slices of brain that have been fixed either before or after slicing into the wells of a 24. Well plate.
Wash the slices in one XPB S3 times for five to 10 minutes per wash. Pipette off the PBS use a paintbrush to center the slice in the well. Place a piece of 3.0 micrometer filter between the two metal diffusion screens at the end of the gene gun's barrel liner.
Hold the gene gun so that the end of the barrel liner is 1.5 centimeters away from the sample. Shoot the beads into the slice with 120 to 180 PSI helium gas pressure. Rinse the slices quickly three times with one XPBS to remove extra dye.
Make sure to keep the shot surface of the slice facing up during all liquid handling steps. Store the slices in PBS for three to six hours to allow the DAI to spread along the dendrites cover with foil. As dai is light sensitive, the slices can be mounted at this stage or subjected to further staining with antibodies as detailed in the next section to proceed with staining at 300 to 500 microliters of permeation solution containing 0.01%TRITTON X 100 in one XPBS to each slice, incubate at room temperature for exactly 15 minutes.
Remove the permeation solution from the wells block the slices in solution containing 10%goat serum and 0.01%tritan X 100 in one XPBS incubate at room temperature for 30 minutes. Add 300 to 500 microliters of primary antibody diluted in blocking solution to each. Well incubate at room temperature for one to two hours or overnight, depending on the antibody wash slices three times with one XPBS for five to 15 minutes each wash, remove the solution from the well.
Add 300 to 500 microliters of secondary antibody diluted in blocking solution to each. Well wash four times with one XPBS for five to 15 minutes each wash as before, mount the slices onto a glass slide with fluorescent mounting medium and cover with an 18 by 18 millimeter. Cover slip allow to dry for six to 12 hours at room temperature before sealing the edges with clear nail polish stored in the dark or covered at four degrees Celsius.
Exemplary images of labeled brain sections are shown here. Two important characteristics to look for are a sparse labeling pattern at low magnification and the ability to identify individual cellular components. Incorrect preparation of the bullets or over fixation of the tissue can lead to bad labeling as shown here.
Troubleshooting tips are provided in the text here. There is a confocal image of a TAL medium spining neuron from mouse brain and its complex pattern of dendritic branching. The optical sectioning achieved when using confocal microscopy allows for isolation of single labeled cells in the tissue section.
High magnification images show dendrite segments from a rodent brain and a non-human primate brain. Note, the high intensity of fluorescent staining achieved with this technique resulting in well-defined dendritic spines when combining ICS with immuno staining. This image shows an example of colocalization of D eye labeling in red with immuno staining for GFP expression in green.
This image corresponds to a sal slice from a transgenic mouse expressing the fluorescent protein GFP under the D one dopamine receptor promoter. After watching this video, you should have a good understanding of how to prepare bullets and use the Jing gun to sparsely liable neurons with fluorescent dyes. This technique allows for the clear visualization of neuronal morphology and for the study of dite branching and spines.
