Animals and serial sectioning of the AVN
Adult Sprague-Dawley (SD) rats (n=6) and adult New Zealand White rabbits (n=5) were used. All animals were obtained from the Laboratory Animal Science Department, the Second Affiliated Hospital of Harbin Medical University, Heilongjiang, PR China. All experimental animal procedures were approved by the Local Ethics Committee of Harbin Medical University Animal Care and Use. Briefly, animals were euthanized by cervical dislocation. The heart was removed from each animal, and the AVN region (Figure 1A) was dissected on ice. The AVN region was embedded in optimal cutting temperature (OCT) compound. Four marker points were drawn on the mold in OCT to mark the position of the AVN before it was snap-frozen by liquid nitrogen. Serial cryosections (6-7 μm) were obtained from the AVN tissue by placing the microtome blade at an angle of 20-30° to the vertical axis (Figure 1B), allowing maximum coverage of the atrium as well as visualization of the His bundle. Once typical characteristics of the penetrating bundle (PB) were identified by Masson’s trichrome staining, serial sections were obtained and grouped in preparation for subsequent staining. Sections were processed up to the level of the coronary sinus structure.