After dissection, the hearts should be kept in PBS on ice until the mounting, or be directly mounted in mounting medium and being snap frozen. The embedded hearts should be kept at −80 °C until the cryosectioning is performed.

For sectioning of the aortic roots, it is recommended to use the method described by Nicoletti et al. [7]. Briefly, the proximal 800 μm of the aortic root is serially sectioned on a cryostat, with each section 10 μm thick. “Point Zero,” where the aortic root begins, is located where the cusps of the aortic valves start to be visible and is found by sectioning the heart in an upstanding position in a caudal-to-cranial direction. From this point, 10 μm thick sections, starting at the 90–880 μm levels (distance from “Point Zero”) are collected to slides following scheme of Fig. 1. The first 90 μm of sections are used to adjust the angle to get all three cusps visible and aligned.

Immerse the slides into fixation reagent for 10 min on ice (see Note 11 ).

Remove the slides from fixation reagent and let them dry, at room temperature, for 1 h.

After fixation, slides should be stored at −20 °C until staining.