Immunochemistry and Imaging

For immunostaining of cornea flat mounts, eyes were removed and an incision was made posterior to the limbus to dissect the corneas including a margin of sclera. The corneas were fixed, permeated, and incubated overnight sequentially with blocking, primary, and secondary antibody solutions as previously described.¹⁷ The primary antibodies included were anti-β III tubulin (Abcam, Cambridge, MA, USA; #18207, 1:1000) and anti-CD31 (EMD Millipore, Billerica, MA, USA; #MAB1398Z, 1:100). Negative controls without primary antibodies and using blocking solution (10% normal donkey serum in 0.1% Triton X-100 PBS solution) and subsequently incubated with fluorescently labeled secondary antibodies were performed on cornea frozen sections and showed no positive signal for β III tubulin or CD45 signal. Incisions were made in each sample in order to obtain whole mounts (four quadrants) prior to mounting in 50% glycerol. Imaging of cornea samples was performed on an Olympus FluoView confocal laser scanning microscope (FV500 v5.0 or FV1200; Olympus, Center Valley, PA, USA). Microscope and software settings were identical for all samples within experiments. The size of the Z-stack generated for cornea flatmounts was 12 slices thick (4.77-μm step size) at 10× magnification. To quantify changes in corneal innervation and vasculature, the MetaMorph offline software (version 7.7.0.0; Molecular Devices, LLC, Sunnyvale, CA, USA) was used to calculate the percent threshold area positive for β III tubulin or CD31 staining on acquired confocal images. This threshold area is defined as the percentage of β III tubulin⁺ or CD31⁺ pixels divided by the total number of pixels in the entire image. For each cornea, a representative image from each quadrant was used for analysis (four images per sample where the visual field included the peripheral limbus toward the center of the cornea proper).¹⁷ For immunostaining of sagittal frozen sections, fixed cornea samples were cryoprotected by overnight incubation with 30% sucrose and then frozen in optimal cutting temperature media. Corneal cryostat sections (16-μm thick) were permeated, blocked, and immunostained with anti-β III tubulin (1:1000) and CD45 (BD Pharmingen, San Jose, CA, USA; #550539, 1:200) antibodies.¹⁷ Confocal imaging of representative areas was performed to generate a Z-stack of eight slices (2.4-μm step size) at 40× magnification and 1.5× zoom.