2.5. Multiple immunofluorescence and confocal microscopy

Rats were deeply anaesthetized and intracardially perfused with 4% paraformaldehyde in PBS. The eyes were postfixed in the same fixative for 24 h at 4°C, cryoprotected with 20% sucrose in PBS, embedded in OCT, and sectioned in a cryostat (Leica Biosystems, Germany). Frozen sections (15 μm thick) were stained with a mixture of primary antibodies (Table 1) overnight at 4°C, exposed to the proper secondary antibodies (Table 1), and counterstained with DAPI (1:1000, Sigma). Images were acquired in a confocal microscope (TCS SP5, Leica).