Fc7 cryochamber

Retinas from blue LED-induced RD mice were fixed with 4% paraformaldehyde in 0.1 M PB for 2 h and transferred to 2.3 M sucrose in 0.1 M PB for 24 h for dehydration. The retinas were then frozen in liquid nitrogen and cut into semi-thin cryosections (2 μm) at −100 °C using a Leica EM UC7 ultramicrotome equipped with an FC7 cryochamber (Leica, Wetzlar, Germany). The sections were blocked with 10% normal donkey serum in PBS for 1 h at room temperature and then labeled at 4 °C overnight using a 1:100 dilution of CD206 antibody. After washing in PBS, the samples were incubated with a secondary donkey anti-rabbit-peroxidase antibody for 1 h (1:100, Sigma). Tissue sections were washed with PBS, followed by rinsing in 0.05 M TB (Tris-HCL, Biosesang, Incheon, Republic of Korea). The sections were then incubated with DAB solution for a few minutes, washed with 0.1 M PB for 10 min, and post-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide for 30 min. Silver enhancement was performed using the HQ silver enhancement kit (Nanoprobes, Yaphank, NY, USA) for 3 min. Sections were dehydrated in graded alcohol and embedded in Epon 812 (Polysciences, Warrington, PA, USA). Areas of interest, selected under light microscopy, were cut into ultrathin sections (80–90 nm) and observed under an electron microscope (JEM 1010, Tokyo, Japan).