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.