Formaldehyde

Nineteen days after ONC, FG (Cambridge Biosciences, Cambridge, UK) was injected into the optic nerve, between the lamina cribrosa and the ONC site (Vigneswara et al., 2013, 2014 ). Animals were killed 2 days later and the dissected retinae were immersion-fixed in 4% formaldehyde (TAAB Laboratories, Aldermaston, UK), flattened onto Superfrost Plus microscope slides (VWR International, Lutterworth, UK) by making four equidistal radial cuts to obtain four quadrants, air dried and mounted in Vectamount (Vector Laboratories). The identity of individual retinae was anonymised by a second investigator before image capture using a Zeiss epifluorescent microscope and the number of FG⁺ RGCs counted in ImagePro Version 6.0 (Media Cybernetics) from captured images of 12 rectangular areas, 3 from each quadrant, placed at radial distances from the centre of the optic disc of the inner (1/6 eccentricity), midperiphery (1/2 eccentricity) and outer retina (5/6 eccentricity). The mean densities of FG⁺ RGCs/mm² for each retina were determined.

All washes and dilutions were performed in Buffer A (PBS pH7.4 containing 5 mg per ml BSA and 1 mg per ml glucose). Cells were seeded on 0.01% (w/v) poly-l-lysine (Sigma-Aldrich) coated coverslips and incubated for 30 min at 37 °C in 5% CO₂. Cells were fixed in 1% formaldehyde (TAAB Laboratories Equipment) and permeabilised in 0.05% saponin (Sigma-Aldrich). After permeabilisation, all subsequent washes and antibody dilutions were carried out in Buffer A containing 0.005% saponin. Secondary antibodies used were goat anti-mouse Alexa Fluor^® 488 and goat anti-rabbit Alexa Fluor^® 546 conjugated antibodies (Invitrogen) diluted in 4% (w/v) normal goat serum. Coverslips were mounted on Vectashield^® Mounting Medium (Vector Laboratories). Samples were imaged at 22 °C using 40x oil immersion lenses (magnification 101.97 µm at zoom 3.8, 1.25 NA) on a Leica DMI 6000 inverted microscope with phase contrast connected to a Leica TCS SP5 confocal imaging system (Leica). Control slides were used to set the fluorescent levels and these were kept to subsequently image the patient samples. Images were obtained using Leica LAS AF software and subsequently processed using Adobe Photoshop (Adobe) and Volocity v.6.3 (Perkin Elmer).