TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Collection_Histology_Microscopy
AUTHORS: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for collection, histological processing, and imaging of rat vagus nerves.</div></div>

[STEPS]
?. [Collect rat vagus nerve samples.]
We collected vagus nerve samples from perfused Sprague-Dawley rats that were used in other experiments approved by the Duke University Institutional Animal Care and Use Committee.
?. [Collect rat vagus nerve samples.]
We perfused the rats with ~300 mL phosphate buffered saline or until the liver was cleared, followed by ~300 mL 4% paraformaldehyde or until the joints and tissues were stiff.
?. [Collect rat vagus nerve samples.]
We collected samples of the carotid sheath in the neck bilaterally, where the vagus nerve courses straight with the common carotid artery, from the clavicle up to where the nerve and carotid intermingle with non-parallel other nerves. We measured from the "valley" of the common carotid bifurcation to the center of each sample.
?. [Collect rat vagus nerve samples.]
We collected the esophagus from the diaphragm to the stomach with the subdiaphragmatic vagus nerves attached.
?. [Collect rat vagus nerve samples.]
We dyed the rostral end of each sample green to maintain orientation during processing.
?. [Collect rat vagus nerve samples.]
We placed each sample between two histologysponges in a mega-sized histology cassette. We placed the cassettes in a tub with 4% paraformaldehyde in a 4oC refrigerator.
?. [Perform histological processing.]
We rinsed each sample with deionized water.
?. [Perform histological processing.]
We processed each sample on the long cycle inthe Leica ASP300S Tissue Processor for ~10 hours: 70, 80, 95, 95, 100, 100, 100% ethanol for 30, 35, 40, 40, 40, 40, 40 min, respectively; Clearite for 50 min, three times; paraffin wax for 50 min, three times.
?. [Perform histological processing.]
We embedded each sample in a paraffin block oriented to obtain transverse sections.
?. [Perform histological processing.]
After trimming to the center of the sample, we collected 5 µm sections, placing two serial sections per microscope slide for fifteen slides.
?. [Perform histological processing.]
The slides were air dried overnight at room temperature, then baked at 37oC overnight.
?. [Perform histological processing.]
Of the 15 slides per sample, we stained slides 2 and 14 with Masson's trichrome as follows.
?. [Perform histological processing.]
The slides were baked at 60oC for 1.5 hours and then cooled overnight.
?. [Perform histological processing.]
We deparaffinized the slides and hydrated them to distilled water: xylene (2x 6 min), 100% ethanol (5 min), 95% ethanol (4 min), 70% ethanol (3 min), dH2O (2x 1 min).
?. [Perform histological processing.]
We placed the slides in Bouin's fixative (mordant) at room temperature overnight.
?. [Perform histological processing.]
We washed the slides in running tap water untilthe yellow color disappeared (~10 minutes).
?. [Perform histological processing.]
We rinsed the slides in distilled water.
?. [Perform histological processing.]
We placed the slides in Weigert's Iron Hematoxylin solution for 10 min.
?. [Perform histological processing.]
We washed the slides in running tap water for 10min.
?. [Perform histological processing.]
We placed the slides in Biebrich Scarlet-Acid Fuchsin solution for 5 min.
?. [Perform histological processing.]
We washed the slides in running tap water for 2min.
?. [Perform histological processing.]
We placed the slides in Phosphomolybdic-Phosphotungstic Acid solution for 10 min.
?. [Perform histological processing.]
We transferred the slides directly to Aniline Blue solution for 3 min.
?. [Perform histological processing.]
We differentiated the counterstain (A. blue) in 1% Acetic Acid solution for 1 min.
?. [Perform histological processing.]
We dehydrated, cleared, and coverslipped the slides: 95% ethanol (1 min), 100% ethanol (4 min), xylene (5 min).
?. [Perform microscopy.]
Each sample was imaged at 20x using a Nikon Ti2 microscope with a Photometrics Prime 95B-25MM camera (Nikon Instruments Inc.). We selected the best of four slices for each sample based on the quality of the slice (no tearing or fraying).