The overall goal of this procedure is to determine the effective treatment on the morphology of neurons within the rodent brain. This method can help answer key questions in the neuroscience field, such as the normal and experimentally manipulated morphology of neurons within various regions of the rodent brain. This technique has the main advantage of increasing the probability of identifying and visualizing labeled neurons that are fully contained within single histological samples.
Begin Golgi-Cox staining by placing a fresh mouse brain into a 20 milliliter glass scintillation vial containing 17 milliliters of Golgi-Cox solution. Protect from light and incubate at room temperature for 25 days. Once the incubation period has elapsed, Cryo protect the brain by placing it into 40 milliliters of sucrose cryoprotectant, and incubate in the dark at 4 degrees celsius for 24 hours.
After the incubation in sucrose, the brain can be frozen for long term storage, or immediately blocked for sectioning as shown in the next section of the video. If freezing, immerse the whole brain in 200 milliliters of isopentane that has been precooled on dry ice. Then place the frozen brain into a 50 milliliter conical tube and store in the dark at minus 80 degrees celsius.
To begin, remove the brain from the sucrose. And use a razor blade to block it for sectioning by cutting off the cerebellum, leaving a flat edge at the remaining caudal end of the brain. The brain must be blocked at a precise angle to ensure that neurons of interest are fully contained within the resulting sections.
For example, we use a coronal plane that is perfectly perpendicular to the rostral caudal axis for cerebral cortex pyramidal neurons. Next, heat agar until it is melted. And let it cool until it is slightly above it's melting point.
Then place the brain in a small disposable weigh dish with the caudal end face down. Add melted agar to cover the brain. Once the agar has solidified, trim the excess leaving approximately two to four milliliters surrounding the brain.
Then use a small amount of ethyl cyanoacrylate to glue the brain to the stage of a vibratome with it's caudal end face down. Fill the stage area of the vibratome with enough sucrose cryoprotectant to cover the brain. Then section the brain at a slice thickness of 400 to 500 microns depending on the brain region to be examined.
Using a small paintbrush, place brain sections into wells of a six well tissue culture plate containing mesh bottom inserts and pre filled with sucrose M phosphate buffer. Protect from light during sectioning. When finished sectioning, incubate sections in the dark at four degrees celsius overnight.
Using the mesh bottom inserts, transfer the brain sections into a new well containing five milliliters of paraformaldehyde in phosphate buffer. Incubate on a rocker moving at slow speed at room temperature in the dark for 15 minutes. Wash the sections twice by transferring them to new wells containing five milliliters of water.
Protect from light and wash at moderate speed at room temperature for five minutes. Next, transfer the sections into a new well containing five milliliters of ammonium hydroxide. Rock slowly in the dark at room temperature for 15 minutes.
Wash the sections twice by transferring them to new wells containing five milliliters of water. Wash on a rocker moving at moderate speed in the dark at room temperature for five minutes. Transfer the sections into a new well containing five milliliters of diluted fixative A.Incubate on a rocker moving at slow speed in the dark at room temperature for 25 minutes.
Wash sections in water twice as before. Use a small paintbrush to place the sections onto a microscope slide, then use tweezers and a small tissue to remove excess water and agar. Ensure that all agar is removed before proceeding with the dehydration steps.
Allow the sections to air dry at room temperature protected from light. The appropriate drying time is critical, and should be determined for each laboratory. Too short of a time may result in sections falling off of slides during dehydration steps, and too long of a time may result in sections cracking.
After drying, first place the slides in clearing agent for five minutes. Place the slides in 100%ethanol for five minutes. Then move through a series of decreasing ethanol concentrations for two minutes each.
After the alcohol series, place the slides in water for five minutes. Then stain in 0.5%cresyl violet in water for seven minutes. Following the cresyl violet stain, place the slides in water for two minutes.
Then dehydrate the sections through a series of increasing alcohol concentrations for two minutes each. Then two washes in 100%ethanol for five minutes. Place in clearing agent for five minutes.
Finally add an anhydrous mounting medium and place a cover slip over the sections. Allow slides to dry horizontally in the dark at room temperature for at least five days. To capture high resolution image stacks of the area containing the neuron of interest, first select a 30X 1.05 NA silicone oil immersion objective on the microscope.
Then apply three to four drops of silicone immersion oil to the slide. Place the objective over the slide, ensuring contact between the objective and oil. Focus the image and adjust the camera settings including the exposure and white balance in the imaging software.
Next set the upper and lower boundaries for the image stack by focusing to the top of the section, and then selecting set next to top of stack within the image acquisition window. Then focus to the bottom of the section, and select set next to bottom of stack. Set the step distance to one micron by entering one micron next to distance between images within the image acquisition window.
Lastly capture the image stack by selecting acquire image stack within the image acquisition window. Repeat the previous steps to capture the entire area of interest making sure that all image stacks overlap by at least 10%in the X and Y axes. This image of the cerebral cortex is a merged Z projection of image stacks acquired from a 500 micron thick section using a 10X objective.
The tissue in this image was processed using the main all fresh protocol. The area indicated by the red square was imaged using a 30X silicon oil immersion objective and a higher resolution merged Z projection image stack was acquired. The red arrow shows the two dimensional Z projection of a neuron that was traced from the high resolution 30X image stack.
Here the fresh protocol variant with cresyl violet staining was used. This higher resolution image shows the cresyl violet stained section. And this is the neuron tracing obtained.
Finally this tissue was processed using the protocol variant in which brains are frozen following Golgi-Cox impregnation. Here the previously frozen tissue is seen at higher resolution. And once again a high quality two dimensional Z projection of the neuron tracing is obtained.
This technique can be completed within one month with the processing and imaging steps completed within the final week. Brains can alternatively be frozen following Golgi-Cox impregnation allowing for the processing and imaging to be completed at a later date. Following this procedure, neuroscientists can use the captured images to perform detailed morphological analyses such as dendrite complexity or spine density in order to determine how an experimental manipulation can alter the structure of neurons within the brain.
