Anti iba1

Exo-green MSC-EV-injected ischemic and normal rats were anesthetized at different time points (1, 3, and 7 days) after intravitreal injections and subjected to whole animal perfusion-fixation with PBS and 4% paraformaldehyde [52 (link)]. Following enucleation, the eye cups were prepared by removing the cornea, lens and vitreous. The eyecups were post-fixed in 4% PFA for 30 min, washed twice in PBS, and permeabilized with PBST (0.3% Triton X-100 in PBS, twice). The eye cups were blocked overnight in 2% Triton X-100, 10% normal serum and 1 mg/ml BSA [53 ]. The primary antibodies anti-IBA-1 for retinal microglia [54 (link)] (1:500, Novus Bio, Littleton, CO), and anti-Brn-3a for retinal ganglion cells [55 (link)](1:500, EMD Millipore), and anti-β-tubulin III for retinal neurons [56 (link)] (1:500, Sigma), were incubated with the eyecups at 4°C for 48 h followed by washing and incubation with the appropriate secondary antibodies (Alexa Fluor 555 and 647, Molecular Probes, Thermo-Fisher) for an additional 48 h at 4°C. The samples were washed again, and the retinal tissues carefully dissected from the choroid and placed on a glass slide and mounted with Pro-Long Diamond Antifade Mounting Solution with DAPI (Life Technologies, Thermo-Fisher). Slides were imaged using a Zeiss 710 confocal at 63 and 100× oil immersion magnification, and images deconvoluted using Zeiss Zen v2.4 software.