Animals were perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer 0.1 M after a saline rinse. Eyes were dissected and cryoprotected in increasing gradients of sucrose (15% to 30%; Sigma-Aldrich, Madrid, Spain). Tissue was embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen, and kept at −80°C. Retinas (n = 4/group) were sectioned at 15 μm in a cryostat. For immunodetection, sections were blocked in 2% donkey serum in phosphate-buffered saline (PBS) with 0.1% Triton (PBST) and incubated overnight at 4°C with a mixture of rabbit antiglial fibrillary acidic protein (GFAP; 1:500 Sigma-Aldrich) and goat anti-vimentin (1:500 sc-7557; Santa Cruz Biotechnologies, Heidelberg, Germany) or a mixture of mouse anticellular retinaldehyde binding protein 1 (CRALBP1 [B2], 1:200, ab15051; Abcam, Cambridge, UK) and goat anti-brain-specific homeobox/POU domain protein 3A (Brn3a, 1:750, sc-sc-31984; Santa Cruz Biotechnologies). Secondary detection was done with donkey anti-goat Alexa 488 or 594, donkey anti-mouse Alexa 488, and donkey anti-rabbit Alexa 594 (1:500; Molecular Probes, ThermoFisher, Madrid, Spain). Finally, sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Vectashield mounting medium with DAPI; Vector Laboratories, Palex Medical, Barcelona, Spain). Images were acquired with a ×20 objective under an epifluorescence microscope (Axioscop 2 Plus; Zeiss Mikroskopie, Jena, Germany).
Brn3a+RGCs were manually counted, in a masked fashion, in sections spanning the optic nerve (3 sections/animal). Numbers are given as the mean number ± standard difference of RGCs per section.