Cryostar nx50 microtome

Porcine corneas were obtained and handled as described above. After excising and 1 h incubation in cornea media, each half cornea was infected with 10⁶ pfu of strain 17 or 17Δγ34.5 mutant virus in 1 mL of media. At 48 hpi, corneas were washed in PBS and embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and stored at −80°C. 10 μm sections were cut using Cryostar NX50 microtome (Thermo Scientific). Sections were air dried at room temperature for 10 min and then fixed in ice-cold acetone for 15 min. Two 10 min washing steps were performed in TBS containing 0.05% Tween 20 (TBS-T) before blocking with 1% BSA in TBS-T for 1 h at room temperature. Sections were then incubated with a cocktail of mouse anti-HPSE (1:100, Santa Cruz Biotechnology sc-293205) and rabbit anti-HSV (1:100, Abcam ab9533) overnight at 4°C. As a control, sections were incubated without primary antibodies. After subsequent washes in TBS-T sections were incubated with a cocktail of anti-mouse Cy5 (1:200, Abcam ab6563) and anti-rabbit FITC (1:200, Sigma F9887) for 1 h at room temperature. Following washes in TBS-T, sections were mounted in Vectashield mounting media containing DAPI (Vector Laboratories, Burlingame, CA) and imaged at 20× magnificiation on LSM 710 confocal microscope (Zeiss).