Image pro software version 6

1 × 10³ HFSCs/well were placed into 6-well culture plates pre-coated for 1 h with Geltrex (n =4). After 14 days of culture, the colonies were stained with hematoxylin (Solarbio). The colony number was determined from scanned images using Image-pro software version 6.0 (Media Cybernetics, Rockville, MD, USA).

RGC were retrogradely labelled with FG (Cambridge Bioscience, Cambridge, UK) by injecting 2 μL of a 4% FG solution into the optic nerve, proximal to the ONT site, 48 h before enucleation [34 (link),35 (link),42 (link),43 (link)]. Retinae (n = 16/group) were immersion-fixed for 2 h in 4% formaldehyde (TAAB Laboratories, Berkshire, UK), adhered onto Superfrost Plus microscope slides (VWR international, Leicestershire, UK) as whole mounts and cover slipped in Vectashield fluorescent mounting medium (Vector Laboratories, Peterborough, UK). Retinae were randomized and examined using an epifluorescent microscope (Zeiss, Hertfordshire, UK) and photographed with an AxioCam HRc digital camera controlled by Axiovision 4 software (all from Zeiss). FG⁺ RGC were counted using the automated function in ImagePro Software, version 6.0 (Media Cybernetics, Bethesda, USA) from images of 12 rectangular areas (0.36 × 0.24 mm), 3 from each quadrant of each retina, placed at standard radial distances from the centre of the optic disc at the inner (1/6 eccentricity), mid-periphery (1/2 eccentricity), and peripheral retina (5/6 eccentricity) and RGC densities computed as mean RGC density/mm² (n = 8 rats/group, 16 retinae/group) [41 (link)].