*2.5. Immunofluorescence Analysis*

Ocular globes were para ffined, sectioned (4 μm) and mounted on poly L-lysine positive charged slides (Leica Biosystems, Nussloch, Germany). The samples were depara ffinized in xylene (VWR, Barcelona, Spain), rehydrated in grade ethanol series (100%, 96%, 70% and 50%), fixed again in ice-cold acid methanol (−20 ◦C) and washed 3 × 5- with phosphate-bu ffered saline 0.01 M (PBS) at pH 7.4. Successively, slides were warmed in a pressure cooker for 4 min at 150 ◦C in 250 mL of antigen retrieval with sodium citrate 10 mM, pH 6. Then, the sections were blocked with blocking solution (protein block serum-free, X0909 Agilent, Santa Clara, CA, USA) for 1 h at room temperature and they were subsequently incubated overnight at 4 ◦C with specific primary antibodies (Table 2). Next day, after 3 × 10- washes in PBS, the samples were incubated for 1 h in darkness with secondary antibodies (Alexa 488 and Alexa 594; 1/600, Molecular Probes, Eugene, OR, USA). The sections were washed again 3 × 5- with PBS, counterstained with Hoechst 33,342 (bisbenzimide) (Thermo Fisher Scientific, Eugene, OR, USA) and mounted with mounting medium fluorescence (Prolong, Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) and coverslips. Images were acquired using laser confocal microscopy (Fluoview FV 1000 laser scanning confocal microscope Olympus, Hamburg, Germany) at a resolution of 1024 × 1024 pixels. Immunofluorescence was quantified with Image J software.




**Table 2.** *Cont.*
