TITLE: Tissue clearing of human cardiac tissues using modified iDISCO+ protocol
AUTHORS: Peter Hanna, Kalyanam Shivkumar, Shumpei Mori, Olujimi Ajijola

[DESCRIPTION]
The purpose of this protocol is to evaluate the myocardial innervation of the human heart. Using donor hearts rejected for human transplantation, imaging of the heart was performed, and sections of the heart underwent tissue clearing and confocal imaging.

[STEPS]
SECTION: Tissue Fixation
1. Donor human hearts are obtained from the organ procurement organization and undergo perfusion fixation.
SECTION: Tissue Fixation
2. Suture three of the pulmonary veins and the inferior vena cava closed. One pulmonary vein and the superior vena cava are left patent as outflow of perfusate.
SECTION: Tissue Fixation
3. Cannulate aortic root and pulmonary artery with 24 Fr cannulas.
SECTION: Tissue Fixation
4. Perfuse 4% paraformaldehyde (PFA) via these cannulas at flow rates to distend the cardiac chambers for 24 hours at 4oC.
SECTION: Tissue Fixation
5. Wash the heart to remove PFA with 0.01 mol/L phosphate buffered saline (PBS) perfusate for 30 minutes via the cannulas followed by three 0.01 mol/L PBS washes for 30 min.
SECTION: Tissue Fixation
6. Store the heart in 0.01 mol/L PBS/0.02% sodium azide at 4oC until processing for immunohistochemistry or modified immunolabeling-enabled three-Dimensional
Imaging of Solvent-Cleared Organs (iDISCO+) tissue clearing.
SECTION: CT imaging, Photography and Dissections of Specimens
7. The heart is suspended in a container and scanned with a 64-multidetector-row computed tomographic scanner (SOMATOM Definition AS, Siemens Healthcare, Forchheim, Germany) with the following parameters: tube voltage, 120 kV; tube current, 800-900  mA; field of view, 100-200 mm; section thickness, 0.6 mm; an incremental  interval, 0.3 mm; matrix size, 512*512, and dose length product, 500-1200 mGy*cm. Postprocedural analysis is performed using commercially available software (Ziostation2; Ziosoft, Tokyo, Japan).
SECTION: CT imaging, Photography and Dissections of Specimens
8. Specimens are dissected using either a #11 surgical scalpel or Metzenbaum scissors to generate adjacent samples intended for immunohistochemistry or tissue clearing using the modified immunolabeling-enabled three-Dimensional Imaging of Solvent-Cleared Organs (iDISCO+) protocol.
SECTION: Tissue Clearing using iDISCO+ Protocol
10. Fixed tissue undergo graded methanol (MeOH) dehydration (20, 40, 60 and 80% MeOH in diH2O [vol/vol]) for 1 hour each at room temperature (RT) with agitation.
SECTION: Tissue Clearing using iDISCO+ Protocol
11. Specimens are washed twice with 100% MeOH for 1 hour at RT and incubated in 66% dichloromethane/33% MeOH overnight at RT with agitation.
SECTION: Tissue Clearing using iDISCO+ Protocol
12. Tissues are then washed twice in 100% MeOH for 1 hour at RT, chilled at 4oC and then bleached in 5% H2O2 in MeOH (vol/vol) overnight at 4oC.
SECTION: Tissue Clearing using iDISCO+ Protocol
13. The tissues are rehydrated with a graded MeOH series and washed in 0.01 M PBS for 1 hour each at RT with agitation.
SECTION: Tissue Clearing using iDISCO+ Protocol
14. The tissues are washed in 0.01 M PBS with 0.2% Triton X-100 for 1 hour at RT.
SECTION: Tissue Clearing using iDISCO+ Protocol
15. Tissues are then stained by permeabilizing in 0.01 M PBS, 0.2% Triton X-100, 20% dimethyl sulfoxide (DMSO) and 0.3 M glycine and blocking in 0.01 M PBS with 0.2% Triton X-100, 10% DMSO and 5% donkey serum, each for 2 days at 37oC with agitation.
SECTION: Tissue Clearing using iDISCO+ Protocol
16. Tissues are then incubated in primary antibodies rabbit anti-protein gene product 9.5 (PGP9.5; Abcam, ab108986, 1:200) and sheep anti-tyrosine hydroxylase (TH; Millipore, AB1542, 1:200) diluted in 0.01 M PBS with 0.2% Tween-20 and 10mg/ml heparin (PTwH) for 1 week at 37oC with agitation to stain. PGP9.5 serves as a pan-neuronal marker while TH serves as a marker of sympathetic neurons. Primary antibodies are replenished mid-week.
SECTION: Tissue Clearing using iDISCO+ Protocol
17. Tissues are washed 4 to 5 times in PTwH overnight at RT and incubated with secondary antibodies donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152, 1:300) and donkey anti-sheep 647 (Jackson ImmunoResearch, 713-605-147, 1:300) for 1 week at 37oC with agitation. Secondary antibodies are replenished mid-week.
SECTION: Tissue Clearing using iDISCO+ Protocol
18. Tissues are again washed in PTwH 4-5 times overnight at RT.
SECTION: Tissue Clearing using iDISCO+ Protocol
19. To clear, tissues are dehydrated with a graded MeOH series as above and incubated in 66% dichloromethane/33% MeOH for 3 hours at RT with agitation.
SECTION: Tissue Clearing using iDISCO+ Protocol
20. Tissues are washed twice in 100% dichloromethane for 15 minutes at RT and cleared, stored, and imaged in benzyl ether (Millipore Sigma, Catalog 108014; refractive index: 1.55).
SECTION: Confocal Imaging of Cleared Tissues
21. Each iDISCO+-cleared tissue is placed in a chamber (SunJin Lab) filled with
benzyl ether on a slide, and a coverslip applied.
SECTION: Confocal Imaging of Cleared Tissues
22. Tilescan and Z stack images are obtained using a Zeiss LSM 880 confocal laser scanning microscope with a Fluar 5x/0.25 M27 Plan-Apochromat and 10x lens.
SECTION: Confocal Imaging of Cleared Tissues
23. Images are obtained at a resolution of 1024 x 1024 using 488, 561, and 633 nm laser lines. Z-axis step size is commensurate with Nyquist sampling based on numerical aperture of the specified objective. Pinhole is set to 1 airy unit.
SECTION: Confocal Imaging of Cleared Tissues
24. Stitched images are analyzed in Zeiss Zen Black SR and Bitplane Imaris 9.5.1
for 3-dimensional visualization.
SECTION: CT imaging, Photography and Dissections of Specimens
9. Hearts are photographed serially during dissection using a Nikon D850 DSLR camera with Nikon AF Micro-NIKKOR 200mm f/4D IF-ED lens.