Supplementary Materials and Methods
β-Galactosidase imunohistochemistry
For immunohistochemical detection of β-galactosidase, eyeballs were collected 5 days after
electrotransfer of pVAX1-LacZ and fixed for 1 hour at room temperature in PBS containing 4%
paraformaldehyde. After washes in PBS, eyes were embedded and frozen in optimal cuttingtemperature (OCT) compound (Tissue-Tek; Sakura Finetek, Zoeterwoude, The Netherlands) and
stored at -80°C. Frozen naso-temporal sections of eyes (10-µm thick) were performed using a
cryostat (CM 3050S; Leica, Rueil-Malmaison, France) and mounted on Superfrost slides (Gerhard
Menzel, Brauschweig, Germany) for immunohistochemical analysis. All following steps were
performed at room temperature. Sections were permeabilized for 30 minutes with 0.1% Triton X-100
in PBS, rinsed with PBS and incubated for 30 minutes with 3% hydrogen peroxide (H2O2) to inhibit
endogenous peroxidase activity. Specimens were rinsed and saturated for 30 minutes with 1%
bovine serum albumin (BSA) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) in PBS. After washing,
they were sequentially incubated for one hour with a rabbit anti-β-galactosidase IgG fraction
antibody (Rockland (200-4136), Tebu-bio, Le Perray en Yvelines Cedex, France) and a biotinylated
goat anti-rabbit IgG antibody (Vector Laboratories (BA-1000), Clinisciences, Montrouge, France)
diluted 1:500 and 1:200 in PBS containing 0.1% BSA, respectively. Omission of the primary antibody
was used in every staining run as a negative control. Sections were then incubated for 30 minutes
with Horseradish Peroxidase-labeled Avidin D (HRP-Avidin D; Vector Laboratories (A-2004),
Clinisciences, Montrouge, France) diluted 1:200 in PBS BSA 0.1%. Peroxidase activity was finally
detected using DAB system (Liquid DAB+ Substrate Chromogen System (K3468); Dako France,
Trappes, France), according to the manufacturer’s instruction, producing a colorimetric brown endproduct at the site of the target antigen. Sections were finally mounted in Fluoromount (SigmaAldrich, Saint-Quentin-Fallavier, France) and observed by light microscopy (Aristoplan light
microscope; Leica, Rueil-Malmaison, France).
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