Lsm 510

Corneal explants were fixed in 4% paraformaldehyde overnight, transferred to 30% sucrose/PBS solution overnight and embedded in OCT compound (Tissue-Tek, Sakura). Ten-micron-thick transversal frozen sections were cut with a cryo-microtome. Sections were stained with 1 μg/ml bis-benzimide Hoechst (Sigma-Aldrich) and mounted with Vectashield (DAKO). For the immunofluorescence CD34 staining, sections were thawed for 15 min at room temperature, washed in PBS for 15 min to remove OCT, permeabilized (0.1% Triton) for 15 min at room temperature and blocked in 2.5% horse serum/0.5% BSA/0.1% Triton for 30 min. Primary antibodies were incubated on sections overnight at 4 °C, and the secondary antibody incubated 90 min, at room temperature prior to Hoechst labeling and mounting. For the human corneas, the primary antibody used was 1:100 mouse anti-human CD34 (clone B1-3C5; AbCam, Cambridge, UK) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa546 (Molecular probes, Invitrogen). For the dog corneas, the primary antibody used was 1:100 mouse anti-dog CD34-PE (clone 1H6, eBioscience) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa Fluor 546 (Molecular probes, Invitrogen). Observations and acquisitions were performed with either a Zeiss LSM510 or a LEICA SP5 laser scanning confocal microscope. Image analyses were performed using MetaMorph software.