TITLE: SPARC_Duke_Grill_OT2-OD025340_VagusNerve_IHC_ChAT
AUTHORS: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes immunohistochemistry with anti-choline acetyltransferase, as it has been applied to cervical and abdominal vagus nerve samples from rats, pigs, and humans.</div></div>

[STEPS]
?. [Immunohistochemistry]
Bake slides with sections of paraffin-embedded vagus nerve overnight at 50oC and then cool overnight.
?. [Immunohistochemistry]
Deparaffinize the slides and hydrate them to distilled water: xylene (2x 6 min), 100% ethanol (5 min), 95% ethanol (4 min), 70% ethanol (3 min), deionized water (2x 1 min).
?. [Immunohistochemistry]
Perform heat-induced epitope retrieval (HIER) at 120oC for 30 s (human) or at 95oC for 5 min (rat, pig) followed by 90oC for 10 s, using a buffer with pH 6.0 (Thermo, TA-135-HBL) for human samples or a buffer with pH 9.0 (BioCare, BD1000MM) for rat/pig samples.
?. [Immunohistochemistry]
Cool for 20 min at room temperature.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Block with 3% H2O2 diluted in deionized water for 10 min.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Block using DAKO Protein Block (X0909) for 10 min at room temperature.
?. [Immunohistochemistry]
Apply the primary antibody (goat anti-choline acetyltransferase, Millipore, AB144P) diluted in Thermo Antibody Diluent to a concentration of 1:100 for cervical & abdominal human samples, 1:50 for cervical pig samples, 1:100 for abdominal pig samples, and 1:200 for cervical & abdominal rat samples. Incubate overnight at 4oC.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply the secondary antibody (biotinylated SP-conjugated Affinipure donkey anti-goat IgG (H+L), Jackson, 705-065-147) diluted in Thermo Antibody Diluent to a concentration of 1:500, and incubate for 1 hour at room temperature.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply ABC Elite (Vector, PK-6100) at a concentration of 1:50 for 30 min at room temperature.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply DAB chromogen (Thermo, TA-125-QHDX) at room temperature for 2.5 min for cervical & abdominal human samples, 2 min for cervical pig samples, 3 min for abdominal pig samples, and 4 min for cervical & abdominal rat samples.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Counterstain using hematoxylin.
?. [Immunohistochemistry]
Dehydrate, clear, and coverslip using DPX mountant.
?. [Microscopy]
Each sample was imaged at 20x using a Nikon Ti2 microscope with a DS-Ri2 color CMOS 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).