A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

The overall goal of this clearing technique is to establish a tissue clearing method for the rat brain. This protocol can also be used for the HIV-1 transgenic rat. The main advantage of this technique is that the rat brain tissue can be left intact for clearing thus allowing for full circuit level analysis.

Demonstrating the procedure will be Kristin Kirchner, a PhD candidate from my laboratory, and also Hailong Li, a research associate from my lab. After performing transcardial perfusion in a three to six week old rat, remove the brain from the skull using forceps. Then place it in a sagittal position and slice it into four equal three millimeter wide sections using a razor blade and a rat brain matrix.

Fix each section in four percent PFA in a sealed five milliliter plastic tube overnight on a shaker at four degrees Celsius. On the next day, continue the fixation at room temperature for one hour. Wash each section three times with PBS for about 30 minutes per wash.

Serially incubate the washed sections in 20, 40, 60, 80, and 100%methanol solution for one hour each. Repeat the dehydration with 100%methanol. Then chill the samples in 100%methanol at four degrees Celsius for 10 minutes.

Prepare a solution containing 66%DCM and 33%methanol and incubate the chilled sections in this solution overnight at room temperature with shaking. On the next day, wash the sample twice with methanol for 30 minutes and chill it in 100%methanol at four degrees Celsius for 10 minutes. For bleaching, place the sample in fresh five percent hydrogen peroxide in methanol and incubate it overnight at four degrees Celsius.

After the overnight incubation, serially incubate the sample in 80, 60, 40, and 20%methanol solutions for one hour each at room temperature. Then incubate the sample in PBS for one hour and wash it twice in solution one for one hour per wash. Fill a one milliliter syringe with 2.5 microliters of one percent biotin-CTB and slowly inject it into the nucleus accumbens site over 20 seconds.

Leave the needle tip in place for one minute and remove it slowly over 10 seconds to prevent leakage. Incubate the sample in solution three and then solution four for two days each in a 37 degrees Celsius water bath. Then add the primary antibody and continue the incubation for seven days.

Wash the sample five times in solution two for one hour per wash and store it in solution two at room temperature overnight. Incubate the sample with a secondary antibody for seven days in a 37 degrees Celsius water bath. Finally, wash it five times in solution two for one hour per wash and store it in solution two at room temperature overnight in low light.

Incubate the sample serially in 20, 40, 60, 80, and 100%methanol for one hour each at room temperature. Then incubate it in fresh 100%methanol overnight. On the next day, incubate the sample in freshly prepared 66%DCM in 33%methanol for three hours at room temperature with shaking.

After three hours, incubate the sample in dibenzyl ether without shaking until it is clear. Then store it in dibenzyl ether until imaging. Obtain a 3D printer chamber and secure it to a microscope slide using epoxy.

Place the sample in the square space in the middle of the chamber and completely fill the chamber with dibenzyl ether. Place a 0.17 millimeter thick cover slip over the chamber and apply pressure to ensure it is in full contact with the chamber walls. Then seal the edges using epoxy.

Make sure to rotate the chamber to allow air bubbles to escape from the filling inlet. Fill the chamber with additional dibenzyl ether. Then seal the filling inlet with epoxy and allow it to cure.

Observe the specimen using a confocal microscope system to perform a z-scan at four times magnification. The confocal image of hydrophobically-cleared tissue sample at four times magnification shows dense TH staining in the substantia nigra area, sparse TH neurons adjacent to the substantia nigra, and a dark black area of tissue lacking TH neurons. Typical morphology of a TH-positive neuron at 20 times magnification has a large soma and several branching processes.

Whereas IBA1-stained microglia have small cell bodies and shorter extending processes. The ideal confocal image shows positive and dense TH staining in the substantia nigra area with suitable fluorescence gain. Examples of poor confocal images include an improperly focused image with too much fluorescent gain, a false positive staining with bright spots not in focus with the rest of the tissue, and a high background staining as a result of using a blocking serum that does not match with a secondary antibody.

Positive CTB staining in the rat brain is shown here. The long thin fibers are afferent projections along the dopaminergic pathway. Colocalization of TH and CTB makes it possible to visualize the injection site in the nucleus accumbens area which appears as a densely fluorescent green circle.

Colocalization of TH and CTB in the substantia nigra is shown here. The most important things to remember in attempting this procedure are to allow the sample plenty of time to incubate in all antibody solutions and plenty of time to fully clear before imaging. The sample should have limited exposure to air as this will damage the sample.

This technique is highly versatile and can be used to investigate many different proteins of interest throughout different regions of the brain. The present technique helps pave the way for neuroscientists to investigate the rat brain on a circuit level which is essential for the study of brain as a whole system.