Immunofluorescent Retinal Labeling Protocol

Flat-mounted retinas were blocked in 100 μL of CTA (5% Chemiblocker, 0.5% TritonX-100, 0.05% Na-azide in PBS) overnight, room temp., humidified. After blocking, the retinas were treated with the primary antibodies (1000× + 1000× mouse SMI31 + SMI32 = SMI312, NE1022/NE1023—Calbiochem; 1000× rabbit Caspase3, AF835—NovusBio; 2000× guinea pig Iba1, 234,004—SySy), diluted in CTA, for 48 h, room temperature (RT). Retinas were incubated for 48 h at RT. After subsequent washing in PBS, three times, secondary antibodies were applied: 500× anti-rabbit Cy3 (715-165-150—Jackson), 1000× anti-mouse A488 (A11017—Invitrogen), and 1000× anti-mouse A647 (A21237—Invitrogen) or anti-guinea pig A647 (A21450—Invitrogen) in CTA, and incubated overnight at room temperature (see Table 1 for antibodies). After washing three times for 10 min in PBS, they were coverslipped with Vectashield (Vector Laboratories, Peterborough, UK) using coverslip nr. 1 (protocol based on and previously validated in [18 (link),61 (link)]).