Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.