Pharmacological Reviews 52, 269C324. also contained nitric oxide synthase or vasoactive intestinal polypeptide. A low-to-moderate density of nerves also stained separately for the latter markers. Additionally, nerve bundles and varicose nerve fibers containing the sensory neuropeptides, calcitonin gene-related polypeptide and substance P, occurred at variable density throughout the ganglia. Collectively, these findings demonstrate that principal neurons of mouse celiac ganglia have less neurochemical diversity than reported for guinea pig and other species but receive input from nerves expressing an array of neurochemical markers. This profile suggests celiac neurons integrate input from many sources to influence target tissues by releasing primarily norepinephrine and NPY. (Eighth Edition, National Academy of Sciences, 2011). 2.2. Tissue Collection and Processing Mice were euthanized using a lethal dose of isoflurane and exsanguinated by cardiac perfusion with phosphate-buffered saline (PBS) containing heparin (1 U/mL). For initial experiments, we used a 10 ml syringe for this step. The celiac ganglia and spleen were removed and immersion-fixed in 4% paraformaldehyde (PFA) in PBS at 4C for 24h, followed by a 24h-incubation in PBS containing 20% sucrose. For most experiment with C57BL/6 mice, we used a peristaltic pump for perfusion at 10 ml/min with 30 mL PBS/heparin followed by 40 ml cold PFA. Dissected tissues were post-fixed overnight in PFA at 4C and then cryoprotected. For sectioning, the cells was freezing with dry snow and inlayed in OCT compound (Ted AMAS Pella, Inc., Redding, CA). Sections of 20C30 m thickness were slice at ?20 to ?25C using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA) and collected on charged slides. Each set of cells sections was boxed separately, wrapped in aluminium foil, and stored at ?20 C until further processing. For collection of belly samples, mice were euthanized and dissected to expose the stomach and intestines. A small incision was made in the proximal belly wall, and the material were flushed with 20 ml PBS via a gavage needle put into the proximal duodenum. This was followed by flushing with chilly PFA, post-fixing over night at 4C and cryoprotection in PBS with sucrose. 2.3. Immunohistochemistry Slide-mounted cells sections were brought to space temp and each was stained for AMAS specific neuronal markers following a protocol of fluorescence immunohistochemistry from earlier studies (Hoard et al., 2008) with minor modification. In summary, slides were AMAS rinsed 4 5 min with PBS (pH 7.3), incubated for 10 min in PBS containing 0.4% Triton X-100 and 0.5% bovine serum albumin (BSA), and blocked for 2 h in PBS containing 1% BSA, 0.4% Triton X-100, and 10% normal donkey serum (Jackson ImmunoResearch IFNGR1 Laboratories, Western Grove, PA, USA). Each section was then double or triple labeled overnight using numerous combinations of main antibodies (Table 1). The following day, the sections were washed 4 5 min with PBS, followed by a 10 min incubation in AMAS PBS comprising 0.4% Triton AMAS X-100 and 0.5% BSA. To reduce background staining, a second blocking step was added before software of secondary antibodies. Species-specific donkey secondary antibodies conjugated to Alexa Fluor 488, 555, 594 or 647 (Jackson ImmunoResearch Laboratories) were applied at a 1:200 dilution in PBS comprising 0.4% Triton X-100 and 1% BSA, and sections were incubated for 2 h before final washing in PBS. After final washes with PBS, cover glasses were applied with Citifluor (Ted Pella, Inc.) or SlowFade Platinum antifade reagent (Existence Technologies Corporation, Eugene, OR, USA) and sealed with clear toenail polish. Specific staining did not occur in bad control sections processed without the addition of the primary antibodies. Table 1. Main Antibodies pii: E1188. doi: 10.3390/ijms19041188. [PMC free article] [PubMed] [CrossRef] [Google Scholar]Berthoud HR, & Powley TL (1993) Characterization of vagal innervation to the rat celiac, suprarenal and mesenteric ganglia. Journal of the Autonomic Nervous System 42, 153C169. [PubMed] [Google Scholar]Berthoud HR, & Powley TL (1996) Connection between parasympathetic and sympathetic nerves in prevertebral ganglia: morphological evidence for vagal efferent innervation of ganglion cells in the rat. Microscopy Study & Technique 35, 80C86. [PubMed] [Google Scholar]Bratton BO, Martelli D, McKinley MJ, Trevaks D, Anderson CR, & McAllen RM (2012), Neural rules of swelling: no neural connection from your vagus to splenic sympathetic neurons. Experimental Physiology 97, 1180C1185. [PubMed] [Google Scholar]Brown TC, Relationship CE, and Hoover DB (2018) Variable manifestation of GFP in different populations of peripheral cholinergic neurons.