This technique provided a more complete and 3D image of entire transparent organs and was useful for quantification, not only of replicating, but also dormant parasite and immune factor cells. This technique allows us to determine the special association of parasites, immune cells, and the tissue damage resulting from the infection. Epitope immunostaining and DNA labeling were also comparable with this technique.
This protocol was effectively used for assessing treatment outcomes in mouse models of Chagas disease. A treatment of higher individual doses, given less frequently over an extended treatment period, eliminates both replicating and dormant parasites. Demonstrating the procedure will be Caleb Hawkins, a research technician from Rick Tarleton's lab.
After euthanizing, dissecting, and fixing the organs of an infected mouse as described in the manuscript, immerse individual organs in 10 milliliters of 50%water-diluted CUBIC-L, with gentle shaking at room temperature in 50-milliliter conical tubes overnight protected from light. Next day, replace 50%CUBIC-L with 10 milliliters of 100%CUBIC-L, and incubate for six days at 37 degrees Celsius, while keeping the tube flat on the shaker incubator. At the end of this incubation period, the tissues should be almost completely transparent.
Wash the transparent organs twice with PBS for two hours at 37 degrees Celsius, with gentle shaking. With each wash, transfer the tissues to a new 50-milliliter conical tube to remove Triton X-100. For DNA staining, dilute the commercially available nucleic acid dye in five milliliters of staining buffer at 1:2, 500.
Then, immerse the tissue in a 15-milliliter conical tube containing nuclear dye solution, and incubate at 37 degrees Celsius, with gentle rotation for five days in a standing position. Wash the tissue three times with 15 milliliters of 3D nuclear staining wash buffer for two hours at room temperature, with gentle shaking. Calculate the required amount of primary and secondary antibodies.
Mix primary and secondary antibodies in an amber two-milliliter tube, and incubate at 37 degrees Celsius for 1.5 hours. For buffer exchange, mix 7.5 milliliters of 2x HV1 3D immunostaining buffer with 7.5 milliliters of double-distilled water. Immerse the tissue sample in the buffer, and incubate for 1.5 hours at 32 degrees Celsius, with gentle shaking in a 15-milliliters conical tube in a horizontal position.
Collect the tissue sample from the buffer exchange media, and immerse it in the antibody staining solution. Incubate tissues individually for one week at 32 degrees Celsius, gently shaking the tubes in a standing position away from the light. Move the tissue samples to four degrees Celsius, and incubate in a standing position.
After cooling the HV1 3D immunostaining wash buffer to four degrees Celsius, wash the sample with 15 milliliters of buffer twice at four degree Celsius for 30 minutes, each with gentle shaking in a horizontal position. Dilute formalin to 1%in HV1 3D immunostaining wash buffer, and immerse the sample in eight milliliters of the solution for 24 hours at four degrees Celsius, with gentle shaking. Now, incubate the sample in fresh 1%formalin solution for one hour at 37 degrees Celsius, and wash in 15 milliliters of PBS for two hours at 25 degrees Celsius.
Immerse transparent organs in a 50-milliliter conical tube containing five milliliters of 50%water diluted CUBIC-R+solution at room temperature, with gentle shaking. Replace it with five milliliters 100%CUBIC-R+and incubate with gentle shaking for two days. Dry the tissues using Kimwipes, and then, transfer them to five milliliters of mounting solution in a six-well culture plate, and incubate them at room temperature.
Adhere the tissues to the microscope sample holder using gel super glue. Immerse the samples in the microscope quartz cuvette filled with 120 milliliters of mounting solution. Image them transversely to their longitudinal axis, and adhere the heart, with the apex and the aorta horizontally aligned.
3D reconstructed Trypanosoma cruzi-infected heart displayed a higher proportion of infected cells in the atrium, compared with ventricles. Dormant parasites can be identified in the heart, as depicted in the 3D-enlarged insets. ZsGreen1-expressing T cells, as well as Trypanosoma cruzi-infected cells, were visualized in the skeletal muscle from a CD8 reporter mouse infected with Trypanosoma cruzi.
3D zoom-ins of 3D-reconstructed image identified T cells in the region of an infected cell. The interface of T cells in host-infected cells was visualized by confocal microscopy. In the heart of an immunodeficient mouse, host cells infected with two different strains of Trypanosoma cruzi were detected, and slicing through the tissues showed abundant parasite-infected cells in the heart atria at various tissue depths.
Immunodetection of vasculature in Trypanosoma cruzi-infected and cleared heart reveals the intricate vasculature of the heart. Parasite-infected cells could be observed in the heart atria. Nuclear staining of skeletal muscle showed an accumulation of nuclei in areas with few or undetectable tdTomato parasites.
Boosting tdTomato and GFP parasite reporter markers with antibodies against RFP, and GFP, and whole cleared skeletal muscle resulted in strong fluorescence. So the critical points are the incubation time in the CUBIC buffer, the dilution and incubation time in the DNA dye, the incubation time in the antibodies, and the incubation time in the refracted index-matching solution. The use of additional staining, with specific antibodies dye and quick reaction, could significantly increase the potential of this technique, allowing for the detection of multiple cells, processes, and structure in intact organs.
We now have a better and more complete way to explore the immune cells responsible for killing the parasites, the tissues where the parasites persist chronically, and the distribution of the specific drugs into the tissues.
