Enhanced 3D Confocal Imaging of Solvent-Cleared Human Heart Tissues

To begin, use a scalpel to dissect 4%paraformaldehyde-fixed human heart tissue to fit within the chamber for confocal microscopy. Dehydrate the heart specimens in a graded methanol series for one hour each at room temperature with agitation, followed by overnight immersion in a 66%dichloromethane and 33%methanol solution with agitation. The next day, bleach the sample by performing the indicated steps.

Then rehydrate the specimen with a graded methanol series for one hour each with agitation. After immunolabeling, perform the following steps to prepare the tissue for imaging. Affix a chamber containing adhesive to a slide and apply nail polish around the perimeter of the chamber.

Turn on the upright laser scanning confocal microscope equipped with a 5X lens. Using laser lines appropriate for the emission spectra of the fluorophores used, acquire Tile scan and z-stack images at a resolution of 1024 by 1024. At the microscale level, tissue clearing of heart samples allows visualization of ganglia containing cardiac neurons and the neural patterning of myocardial innervation.