Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

The human brain is a complex organ spanning different scales. To understand its functionality, it's essential to construct a detailed consensus across the whole brain. Our routinely high throughput protocol permits the analysis of treating architecture of volumetric carriers of the human brain with micrometer resolution, enabling its structural characterization.

Volumetric reconstruction of large areas of the human brain present several experimental challenges linked to the massive dimensions of brain samples, the complex biological composition, the variable postmortem fixation and storage conditions, and the autofluorescent signals from lipofuscin type pigments. All these can compromise the optical clearing efficiency and demonstrating quality. Compared to other clearing techniques, the short tissue transformation method combined with live sheet microscopy imaging can be used for rapid and simultaneous processing of multiple human brain sections.

These result in 3D brain reconstruction with a subcellular resolution. To begin, take a beaker containing PBS and add agarose to prepare a 4%agarose solution. Embed the formalin-fixed human brain tissue block in the agarose solution.

Let it cool to room temperature and store it at four degrees Celsius for 24 hours. For precise sectioning, attach the sample to the vibratome's specimen disc and fill the tray with cold PBS. Adjust the frequency, amplitude, and speed of the vibratome based on the tissue type to be sliced.

After cutting, transfer all the slices into a preservative solution composed of PBS with 0.01%sodium azide. Begin by placing the formalin-fixed paraffin-embedded brain sections in dishes with screw lids. Attach the switch off solution to the dishes, cover them with aluminum foil and incubate at four degrees Celsius while shaking for one day.

The following day, transfer all the samples to new dishes and incubate them with the switch on solution as shown earlier. At the end of the incubation, place the samples into tubes and wash them three times with PBS for two hours while shaking. Then add inactivation solution to the samples and incubate overnight in a water bath set at 37 degrees Celsius.

After performing three washes, add clearing solution to the samples and incubate in a 55 degree Celsius water bath for three to seven days. At the end of the incubation, wash the samples with PBST at 37 degrees Celsius for three hours in an incubator. The next day, add 10 milliliters of 30%hydrogen peroxide to each sample, and wash the samples three times with PBS for 10 minutes while shaking at room temperature.

Next, preheat the antigen retrieval solution to 95 degrees Celsius in a water bath. Transfer the samples into the heated solution and incubate at 95 degrees Celsius for 10 minutes, followed by 40 minutes at room temperature on a shaker. Wash the samples with deionized water for five minutes while shaking.

Equilibrate the samples in PBS. Now, transfer the samples to dishes with screw lids. After preparing fresh primary antibody solution for all the samples in a beaker, add it to the samples and incubate at 37 degrees Celsius for one to seven days with gentle shaking in the dark.

Then, place the samples in tubes and wash them using prewarmed PBST at 37 degrees Celsius in the incubator. Move the samples to dishes with screw lids and add secondary antibodies prepared in PBST and 0.01%sodium azide. Transfer the samples to tubes and wash them with prewarmed PBST three times at 37 degrees Celsius for two hours while shaking.

For refractive index matching, place the samples in new dishes and add 30%solution of TDE. Then replace 30%TDE with 68%TDE solution in the samples, and leave them at room temperature while shaking. After the short processing, optimal and homogenous transparency of all the tissue slices was achieved in both gray and white matter.

To begin, take a brain tissue slide prepared using the short tissue transformation technique. Then place a steel spacer onto the microscope slide and position the sample onto the microscope slide. Carefully position the quartz cover slip over the sample and secure all components together.

Now prepare two component silicon glue mixture in an equal ratio. Using a one milliliter syringe, fill the space between the spacer and the cover slip with the two component silicon glue. Remove the clips holding the assembly together.

Using a small needle, slowly fill the entire sandwich with 68%TDE and seal the sandwich with freshly prepared glue. After imaging the slides, use a blade to gently open the sandwich. Transfer the specimens to new dishes with screw lids filled with a 30%TDE solution for three hours.

Then transfer the samples to tubes and wash them with PBS three times for two hours at room temperature with gentle shaking. Place the samples in clearing solution and incubate them in a water bath set at 80 degrees Celsius for four hours. After incubating the samples, wash them with PBS as shown earlier.

The human Broca's area co labeled for NeuN, somatostatin, and showed two different subpopulations of interneurons. Light sheet fluorescence microscopy images of multi round staining and short processed slices showed white and gray matter labeled with various neuronal markers. Maximum intensity projection images of 500 micrometer thick slice showed the signal from seven different antibodies used in three sequential rounds of immunostaining.