The overall goal for this procedure for cortex flattening and staining is to visualize cellular modules in the cortical regions in a layer-wise manner. This method can answer key questions in neuroscience, like how cortical structure spreads into the brain, and how it might relate to function. The main advantage of this technicque is you can compare cortical architecture in a laminal fashion, and compare various aspects of it, like how it's conserved the class evolution, or how it varies in function.
Demonstrating the technique will be Simon Lauer, and Undine Schneeweib, a student and a technician from the lab of Professor Michelle Black. To begin, transcardially perfuse the animal to obtain a homogeneously fixed and blood-free brain. After removing the brain, submerge it in 0.1 molar phosphate buffer.
If sections with large cortical areas are desired, flatten the brain prior to further postfixation. However, to obtain sections of harder-to-reach areas, like the medial entorhinal cortex, postfix the brain in 4%PFA for 24 hours before flattening. Prepare to keep the brain moist with buffer during the whole dissection.
To begin, separate the hemispheres using a scalpel. If the brain has not been postfixed, be careful when handling it. Next, while securing the brain by the cerebellum, gently insert the spatula to open a dissection plane in the corpus callosum.
The round tip of spatula should point away from the cortex. Then, manipulate the spatula to divide the thalamus and cortex. Next, separate the subcortical structures using a razor blade.
Next, separate the subcortical structures using a scalpel or the edge of the spatula. To improve the flattening, make a clean cut through the striatum, nucleus accumbens, and orbital frontal cortex, since they increase the thickness of the cortex in the lateral region. Then add a relieving cut at the base of the inner region of prefrontal cortex.
This will allow for unfolding of medial portions of the cortex. Now, place a hemisphere cortex down on a glass slide. Then, place two clay cylinders on either side of the tissue.
The clay must a 10 to 20%thinner than the brain tissue, as the clay defines the extent of flattening. Next, place a glass side tangentially onto the cortex, contacting the lateral portion first. Then, set a weight on the glass, with the mass centered over the lateral portion.
Now, let the assembly sit for three to five hours in phosphate buffer at four degrees Celsius. The real key to this preparation is to get the brain as flat as possible, as this has the greatest impact on viewing the cortical map. Lastly, if needed, postfix the brain.
Transfer the flattened hemisphere to 1%PFA, or 2%PFA if it was not taken from a perfused animal. Then, let it fix for 24 hour at 4 degrees Celsius, with gentle agitation. First, wash the flattened hemisphere in phosphate buffer for 15 minutes.
Then, slice the hemisphere tangentially on a Vibratome. Fix the tissue in position using a small volume of cyanoacrylate and gentle pressing. Be careful, as too much glue can lead to cutting artifacts.
Now, cut the hemisphere from the thicker and more stable end towards the thinner end at the required thickness. If low fixative concentrations were used, cut the tissue slowly with a high amplitude. Next, wash the sectioned tissue in hepes buffer for 15 minutes with gentle agitation.
During the wash, prepare fresh cytochrome oxidase staining solution. And add the DAB component just before using it. Now, incubate the sections in the freshly made staining solution at room temperature with shaker and keep watch.
Depending on the amount of fixation, staining could be observable in 10 minutes or it make take several hours. If the reaction is going too slow, increase the temperature to 37 degrees Celsius. To stop the reaction, transfer the sections to a bath of 4%PFA and let them incubate for 15 minutes with gentle agitation.
Then, wash the sections three times, with hepes buffer for 10 minutes per wash. Now, mount and dry the sections on glass slides. And dehydrate them using progressively stronger ethanol baths.
Then, wash the slides in Isopropanol for five minutes, followed by Xylene for five minutes. After the Xylene bath, immediately add a quick-hardening, non water-based mounting medium. Water-based mediums will degrade the stain.
After adding a cover slip, store the slides at four degrees Celsius until imaged. Cortical sections of the somatosensory cortex in a variety of brain were processed, using the described procedures. This comparative approach allows for the study of the evolutionary forces that shape cortex, such as the highly conserved representation of mistacial vibrissaer in rodents and lagomorpha as barrels.
The architecture of the medial entorhinal cortex was observed sections parallel to the peel surface, using tangential sectioning of the medical entorhinal cortex in mice, rats, and Egyptian fruit bats. In addition, human brains were processed using gentle flattening of the cortex and tangential slices. All of the brains were cryopreserved, and sectioned on a cryostat at 60 microns.
Immunostaining was used to label calbindin-positive parameatal cell modules. In the entorhinal cortex the calbindin modules show a remarkable periodicity in each species, and vary in size by only a factor of 10, across a 20, 000-fold variation in brain size. After watching this video you should have a good understanding of how to obtain tangential sections for analysis.
Once mastered, this technique can be completed in a couple of hours. While attempting this procedure, it's important to remember to flatten the brain properly, instead the size, the quality, or the final results. Don't forget that working with the agents like PFA and DAB can be extremely hazardous, and you should always wear protective clothing and work under the field light.
