Immunoperoxidase detection of neuronal antigens in full-thickness whole mount preparations of hollow organs and thick sections of central nervous tissue

• Published in: Journal of neuroscience methods 2011-03, Vol.196 (1), p.1-11
• Main Authors: Llewellyn-Smith, Ida J., Gnanamanickam, Greta J.E.
• Format: Article
• Language: English
• Subjects: Animals; Antigens - metabolism; Bladder; Epoxy Resins; Female; Histological Techniques - instrumentation; Histological Techniques - methods; Immunocytochemistry; Immunoenzyme Techniques - instrumentation; Immunoenzyme Techniques - methods; Medulla; Nerve Tissue - cytology; Nerve Tissue - metabolism; Nerve Tissue Proteins - metabolism; Neurons - metabolism; Rats; Rats, Sprague-Dawley; Rectum; Rectum - cytology; Rectum - metabolism; Uterus; Uterus - cytology; Uterus - metabolism; Whole mount preparation
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2010.12.004

▶ The complete innervation of entire hollow organs visualized in whole mounts. ▶ Morphology of neurons demonstrated in 300 μm-thick sections of brain. ▶ Stained specimens permanent and non-fading. ▶ Required equipment readily available and inexpensive. Immunofluorescently stained whole mounts have proved useful for defining the innervation of the gut and large blood vessels. Nerves supplying other hollow organs are usually studied in sections, which provide much less information. Aiming to describe the entire innervation of rat uterus, we developed a method for immunoperoxidase staining of full-thickness whole mounts that allowed us to visualize all immunoreactive axons. Uterine horns were dissected out, slit open, stretched, pinned flat and fixed. Entire horns were treated with methanol/peroxide, buffered Triton X-100 and normal serum and then incubated in primary antibodies, biotinylated secondary antibodies and avidin-horseradish peroxidase (HRP), each for at least 3 days. Peroxidase reactions revealed immunoreactivity. Immunostained horns were dehydrated, infiltrated with epoxy resin, mounted on slides under Aclar coverslips and polymerized. We treated bladders, gut, major pelvic ganglia and thick sections of perfused medulla oblongata similarly to assess the applicability of the method. Using this method, we could map the entire uterine innervation provided by axons immunoreactive for a variety of antigens. We could also assess the entire tyrosine hydroxylase-immunoreactive innervation in all layers of bladder, gut and ganglia whole mounts and throughout 300 μm sections of medulla. These observations show that this method for immunoperoxidase staining reliably reveals the complete innervation of full-thickness whole mounts of hollow organs and thick sections of central nervous tissue. The method has several advantages. The resin-embedded tissue does not degrade; the immunostaining is non-fading and permanent and neurochemically defined features can be mapped at large scale without confocal microscopy.