The overall goal of this procedure is to reveal complex patterns of gene expression in a whole intact brain structure. This is accomplished by first perfusing the animal and removing the brain. The cerebellum is then dissected and processed using the whole mount staining protocol.
The final step is to visualize gene expression using a histochemical color reaction. Ultimately, bright filled microscopy is used to image surface gene expression patterns. The main advantage of this technique over existing methods like tissue section immunohistochemistry, is the ability to visualize complex patterns in three dimensions without the need for reconstruction.
Demonstrating the procedure is Joshua White, a graduate student from my laboratory. After sacrificing the animal and removing the brain from the skull, separate the cerebellum from the rest of the brain by first inserting the tips of a pair of forceps into the middle of the superior and inferior calli. Then using a second pair of forceps, slowly tease the calli and cortex away from the cerebellum.
Orient the cerebellum so that the anterior side is facing down and the brainstem is pointing upwards. Now insert the forceps into the fourth ventricle between the brainstem and labials nine and 10 of the cerebellum. Cut the peduncles by pinching with the forceps on both sides, and then gently lift the cerebellum away from the brainstem.
Postfix the cerebellum by immersion in 4%paraldehyde at four degrees Celsius from minimum of 24 to 48 hours. The cerebellum can be stored in 4%paraldehyde for an extended period of time While working in a fume hood. Gently pour out the paraldehyde solution and replace with dense fix.
Place the tube containing the cerebellum and fixative solution onto a mutator and rock gently for six to eight hours. After the incubation time has elapsed, pour off the dense fix and replace with dense bleach. Incubate the cerebellum in this solution overnight at four degrees Celsius with rocking the next day.
Replace the solution in the tube with 100%methanol. Place the tube on the mutator and rock gently at room temperature for 30 minutes. After the incubation time has elapsed, replace the solution with fresh 100%methanol and repeat the incubation to ensure dehydration of the cerebellum.
Now, freeze thaw the tissue in 100%methanol by placing the tube in a container with dry ice and freeze for 30 minutes. After this time, use forceps to remove the tube from the dry ice and then place on the bench top to thaw for 15 minutes. Repeat the freeze thaw procedure at least four times to enhance the penetration of the staining solutions.
After the final thaw, place the tube into a minus 80 degrees Celsius freezer overnight. The tissue can be stored for prolonged periods of time at minus 80 degrees Celsius, either immediately before or after the freeze-thaw steps. Otherwise, the protocol should be continued.
The next day. Rehydrate the cerebellum by immersion with rocking in 50%methanol 50%PBS 15%methanol, 85%PBS, and then 100%PBS for 60 to 90 minutes each at room temperature. Next, for adult mouse cerebellar, subject the tissue to enzymatic digestion with 10 micrograms per milliliter proteinase K in PBS for two to three minutes at room temperature with rocking.
This digestion step is not necessary for cerebellar of P five or younger mice. After digestion, immediately pour off the proteinase K and wash the cerebellum in PB S3 times for 10 minutes each to prevent over digestion. After the final wash, replace the solution with P-B-S-M-T and ROCK overnight four degrees Celsius to block the tissue the following day.
Pour off the P-B-S-M-T blocking solution and incubate the tissue in primary antibody diluted in P-B-S-M-T. Supplemented with 5%DMSO for 48 hours at four degrees Celsius with rocking. After the incubation time has elapsed, wash the cerebellum two to three times for two to three hours each in P-B-S-M-T at four degrees Celsius with rocking to remove unbound primary antibodies.
Then incubate the tissue in secondary antibody in a solution of P-B-S-M-T with 5%DMSO at four degrees Celsius overnight. With rocking the next day, wash the cerebellum in P-B-S-M-T as before. Then perform a final wash in PBT for one to two hours with rocking at four degrees Celsius.
This step removes excess milk and enhances the clarity of the staining. Finally, incubate the tissue in DAB solution. Until the optimal staining intensity is reached, it is best to use DAB in the dark.
As light causes the chromogen to precipitate, which can increase background staining. The brown reaction product should be clearly visible within 10 minutes when the optimal staining intensity is reached. Stop the reaction by placing the cerebellum in PBS with 0.4%sodium azide.
The tissue may be stored long term in this solution. Whole mount stain tissue should be imaged while immersed in PBS or another medium that will give an optimal refractive index. In order to easily position the whole mount for imaging, make a 1%agar gel in a plastic Petri dish.
Next, cut small holes in the gel and then wedge the cerebellar into these holes in the desired orientation for imaging cover the cerebellar with PBS. The stained tissue is now ready to be imaged using light microscopy. ZEIN two Adelaide c.
Immunohistochemistry reveals sagittal bands in transverse sections of the cerebellum. Labials four, five, and three are labeled in the diagram. For orientation purposes, the scale bar represents 500 microns.
ZEIN two. Adelaide's C is expressed exclusively in subsets of kinji cell sata and dendrites. In this image, the scale bar represents 150 microns.
This image shows a whole mount cerebellum stained for zein two, Adela C showing bikini cell stripes in images of the anterior, central and posterior zones of the cerebellum. The Roman numerals denote the cerebellar loles. LS is the loist simplex.
PML stands for the paramedian labial. COP denotes the copular parameters and the labels. Crust one and crust two denote the names of two hemisphere LOEs, P one and P three designations are based on a standard nomenclature used to indicate specific BR two stripes.
For further details, Sillitoe and Hawks 2002. The scale bar represents one millimeter. Here the cerebellum has been immunostain with an antibody to cocaine and amphetamine regulated transcript peptide.
It can be seen. That cart is expressed in subsets of climbing fibers that travel through the bikini cell layer labeled PCL and terminate in the molecular layer labeled ml. Here, the scale bar represents 100 microns.
It can be seen in this image that the cart positive fibers project a stripes of bikini cell dendrites in the molecular layer of the cerebellar cortex, including regions such as the Oculus labeled PFL, and the Oculus labeled fl. While attempting this procedure, it's important to remember to avoid nicking the cerebellum. Any external damage will obscure the visibility of complex staining patterns.
After watching this video, you should have a good understanding of how to reveal complex and interesting patterns of gene expression in neurons and in the fibers that contact them in the intact brain.
