Corneas were dissected and fixed at room temperature for 1 hour in 1.3% paraformaldehyde in PBS, and radial incisions were made to facilitate flat-mounting of the corneal tissues. Corneas were washed in PBS 5 times, permeabilized in 1% Triton X-100 in PBS at room temperature for 60 minutes and blocked with 20% goat serum (Cedarlane, Burlington, NC, USA) in blocking buffer (0.3% Triton X-100/0.1% Tween-20 in PBS) for 1 hour. The corneas were then incubated in a 100 µL cocktail of primary antibodies at room temperature for 2 hours, followed by an additional incubation overnight at 4°C. After five 5-minute washes in wash buffer (0.1% Tween-20 in PBS), the corneas were incubated in a 100 µL cocktail of secondary antibodies in blocking buffer at room temperature for 2 hours. Following five 10-minute washes with wash buffer, the corneas were mounted on slides and dried at 4°C for at least 12 hours before imaging.
Primary antibodies were: rabbit polyclonal anti-Beta III tubulin (1:1000, cat #ab18207), chicken polyclonal anti-tyrosine hydroxylase (TH, 1:200, cat #76442), all from Abcam, Cambridge, MA, USA; or rat anti-substance P (anti-SP, 1:300, cat #4311672, BD Bioscience, San Jose, CA, USA), HSV type-1 antibody, biotin conjugate (Thermo Scientific Prod #PA1-26169 Lot #RB2160088). Secondary antibodies included: Alex Fluor 488 goat anti-rabbit IgG (H + L) (1:500, cat #GR233725-3, Abcam, Cambridge, MA, USA); Alexa Fluor 546 goat anti-chicken IgG (H + L) (1:500, cat #1618409, Life Technologies, Grand Island, NY, USA); Alexa Fluor 633 goat anti-rat IgG (H + L) (1:500, cat #73B1-1) from Molecular Probes, Eugene, OR, USA; and BV421 Streptavidin and 4'-6-diamidino-2-phenylindole (DAPI 1:5000, Sigma, St. Louis, MO, USA).