TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_FibronectinIF_Morphology
AUTHORS: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, 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]
?. [Immunofluorescence]
Bake slides with sections of paraffin-embedded vagus nerve overnight at 50oC and then cool overnight.
?. [Immunofluorescence]
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).
?. [Immunofluorescence]
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).
?. [Microscopy]
Each sample was imaged at 20x (Plan Apochromat Lambda, NA: 0.75) with a GFP/FITC/cy2 filter set (excitation: 466/40 nm (446-486 nm), emission: 525/50 nm (500-550 nm), dichroic mirror: 495 nm; Nikon Instruments Inc.), a SOLA SE II 365 light engine (Lumencor, Beaverton, OR), and a Photometrics Prime 95B-25MM camera (Teledyne Photometrics, Tucson, AZ). 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 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 paired the interior and exterior perineurium traces for a given fascicle.
?. [Image Analysis]
We scaled the pixel coordinates to microns using the segmented scale bar.
?. [Image Analysis]
We calculated the effective diameter of each inner perineurium trace and outer perineurium traces using Matlab’s polyarea, where effective diameter 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.
?. [Immunofluorescence]
Incubate in 0.1% (v/v) Triton-X in PBS for 20 min.
?. [Immunofluorescence]
Incubate in 7.5% (w/v) bovine serum albumin (BSA) in PBS for 1 hour.
?. [Immunofluorescence]
Rinse with 1% (w/v) BSA in PBS.
?. [Immunofluorescence]
Apply the primary antibody (rabbit anti-fibronectin, Sigma, F3648) diluted in 1% (w/v) BSA in PBS to a concentration of 1:50, and incubate overnight at 4oC.
?. [Immunofluorescence]
Rinse with PBS (3x 5 min).
?. [Immunofluorescence]
Apply the secondary antibody (goat polyclonal secondary antibody to rabbit IgG (H+L, FITC) (Abcam, ab97050) diluted in 1% (v/v) BSA in PBS to a concentration of 1:100, and incubate for 1 hour at room temperature.
?. [Immunofluorescence]
Rinse in PBS (3x 5 min).
?. [Immunofluorescence]
Coverslip using Fluoro-Gel II with DAPI (EMS, 17985-50).
?. [Image Segmentation]
We used Nikon's NIS Elements software (v5.02.01, Build 1270) to segment the inner and outer perineurium boundaries using the General Analysis tool. We only segmented fascicles with a single inner perineurium boundary for each outer boundary.
?. [Image Segmentation]
For each image, we selected preprocessing steps, such as smoothing and sharpening.
?. [Image Segmentation]
For each image, we selected fluorescent intensity values to identify the perineurium.
?. [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.