TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_HumanVagusNerve_Claudin1IHC_Morphology
AUTHORS: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Jake E. Cariello, Kara A. Clissold, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes immunohistochemistry with anti-claudin-1, imaging, image segmentation, and image analysis methods to quantify human vagus nerve morphology.</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 followed by 90oC for 10 s, using a buffer with pH 6.0 (Thermo, TA-135-HBL).
?. [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 (rabbit anti-claudin-1, Abcam, ab15098) diluted in Thermo Antibody Diluent to a concentration of 1:50, and 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 goat anti-rabbit IgG (H+L), Jackson, 111-065-144) 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) for 1 min at room temperature.
?. [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 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).
?. [Image Segmentation]
We used Nikon's NIS Elements software (v5.02.01, Build 1270) to segment human vagus nerve immunohistochemical micrographs (anti-claudin-1) using the General Analysis RGB tool.
?. [Image Segmentation]
For each image, we selected preprocessing steps, such as smoothing and sharpening.
?. [Image Segmentation]
For each image, we selected ranges of hues, saturations, and intensities to values that identify the perineurium and different values to identify the entirety of the nerve.
?. [Image Segmentation]
For each image, we selected postprocessing steps, such as setting a minimum size criterion (eliminate small off-target regions), smoothing, cleaning, closing, and filling holes.
?. [Image Segmentation]
We made manual adjustments as needed, including manual deletion of off-target regions and filling of target areas that had not been captured.
?. [Image Segmentation]
We converted the binary segmented image into “Graticule Masks”, binary images saved as TIFs.
?. [Image Analysis]
We imported the TIFs into Matlab and generated a data structure of the x and y coordinates of the pixels for each closed boundary of the loaded binary images using the bwboundaries function.
?. [Image Analysis]
We stored the pixel coordinates with indexing that assigns a fascicle number, which were then checked so that both the interior and exterior perineurium trace relate to the same fascicle.
?. [Image Analysis]
We scaled the pixel coordinates to microns using the segmented scale bar.
?. [Image Analysis]
We calculated cross-sectional area of each fascicle (inner perineurium and outer perineurium traces) and nerve using Matlab’s polyarea. Effective diameter (for a nerve or fascicle) is the diameter of the circle that has the same cross-sectional area as the raw trace. The perineurium thickness is half of the difference in effective diameters of the inner and outer perineurium traces.