Tris hcl

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).

tEVs were isolated from serum using ExoQuick solution (System Bioscience, Palo Alto, CA, USA) with some modifications to the manufacturer’s instructions. After centrifugation to remove cell debris, the serum of six animals in each group were pooled. A total of 2 mL of serum and 500 μL of solution were mixed. After the mixture was centrifuged, the pellet was resuspended in 200 μL phosphate-buffered saline (PBS).
nEVs were isolated as described by Mustapic et al. [52 (link)] with some modifications. Exosomes isolated from serum were incubated with anti-CD171 (L1CAM; Bioss Antibodies, Beijing, China) antibody for 1 h at 4 °C on a rotating mixer. After adding Pierce Streptavidin Plus Ultralink Resin (Thermo Fisher Scientific, Waltham, USA) and PBS, the samples were again incubated for 1 h at 4 °C on a rotating mixer. The samples were then pelleted by centrifugation at 200× g for 10 min at 4 °C. The supernatants were removed from the samples and the pellets were resuspended in 200 μL 0.1-M glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 s and vortexing for 30 s, the samples were pelleted by centrifugation at 4500× g for 10 min at 4 °C. Finally, the supernatants were transferred to new tubes before Tris-HCl (Biosesang) and PBS were added.