TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_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 (paraffin embedding, Masson's trichrome staining), and imaging of pig vagus nerves.</div></div>

[STEPS]
?. [Collect pig vagus nerve samples.]
We collected fresh vagus nerve samples from Yorkshire pigs after they were euthanized following medical training courses at Duke University.
?. [Collect pig vagus nerve samples.]
We collected samples of the carotid sheath in the neck bilaterally. We collected 2 cm samples at the level midway along the rostocaudal line from the rostral end of the sternum to the line connecting the left and right angles of the mandible (typically ~12 to 14 cm apart). We measured from the "valley" of the common carotid bifurcation to the center of each sample.
?. [Collect pig vagus nerve samples.]
We collected ~2 cm samples of the anterior and posterior subdiaphragmatic vagus nerve trunks along the esophagus approximately halfway between the diaphragmatic esophageal hiatus and the gastroesophageal junction.
?. [Collect pig vagus nerve samples.]
We dyed the rostral end of each sample green to maintain orientation during processing.
?. [Collect pig vagus nerve samples.]
We placed each sample between two histology sponges in a mega-sized histology cassette. We placed the cassettes in a tub with 4% paraformaldehyde in a 4oC refrigerator.
?. b.We perfused the rats with ~300 mL 1x PBS oruntil the liver was cleared, followed by ~300 mL 4% paraformaldehyde or untilthe joints and tissues were stiff.
?. c.We collected samples of the carotid sheath inthe neck bilaterally, where the vagus nerve courses straight with the commoncarotid artery, from the clavicle up to where the nerve and carotid interminglewith non-parallel other nerves. We measured from the "valley" of thecommon carotid bifurcation to the center of each sample.
?. d.We collected the esophagus from the diaphragm tothe stomach with the subdiaphragmatic vagus nerves attached.
?. e.We dyed the rostral end of each sample green tomaintain orientation during processing.
?. f.We placed each sample between two histologysponges in a mega-sized histology cassette. We placed the cassettes in a tubwith 4% paraformaldehyde in a 4oC fridge.
?. [Perform histological processing.]
Each sample underwent dehydration, etc.processing.
?. [Perform histological processing.]
Each sample was cut in half along its length andembedded in a single paraffin block with the two central surfaces of the nerveexposed for the initial slices.
?. [Perform histological processing.]
5 µmslices were taken and placed on microscope slides.[NP1]The slides were stained with Masson’s trichrome.
?. [Perform histological processing.]
0pt'>[NP1]Couldprovide more details on the slicing (took 30 slices, mounted on slides in thisorder, stained slides ## and ## with MT, …), but perhaps too much detail?
?. [Perform histological processing.]
?. [Collect rat vagus nerve samples.]
We perfused the rats with ~300 mL 1x PBS oruntil the liver was cleared, followed by ~300 mL 4% paraformaldehyde or untilthe joints and tissues were stiff.
?. [Collect rat vagus nerve samples.]
We collected samples of the carotid sheath inthe neck bilaterally, where the vagus nerve courses straight with the commoncarotid artery, from the clavicle up to where the nerve and carotid interminglewith non-parallel other nerves. We measured from the "valley" of thecommon carotid bifurcation to the center of each sample.
?. [Collect rat vagus nerve samples.]
We collected the esophagus from the diaphragm tothe stomach with the subdiaphragmatic vagus nerves attached.
?. [Collect rat vagus nerve samples.]
We dyed the rostral end of each sample green tomaintain orientation during processing.We placed each sample between two histologysponges in a mega-sized histology cassette. We placed the cassettes in a tubwith 4% paraformaldehyde in a 4oC fridge.
?. [Perform microscopy.]
Each sample was imaged at 10x 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).
?. [Perform histological processing.]
We rinsed each sample with deionized water.
?. [Perform histological processing.]
We processed each sample on the long cycle in the 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 50min, three times; paraffin wax for 50 min, three times.
?. [Perform histological processing.]
We cut each sample in half transversely and embedded the halves together cut side down in paraffin in order to obtain transverse sections starting from the center of the nerve and moving outward.
?. [Perform histological processing.]
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 until the 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 2 min.
?. [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).