Imaging ICAM-2 Expression in Laser-Induced CNV

Eyes were treated as previously described^{6 (link),11} . Briefly, mice were sacrificed 2 weeks after laser-induced CNV. Enucleated eyes were fixed for 1 h in 1% paraformaldehyde (#15,713-S, Electron Microscopy Sciences, Hatfield, PA) at room temperature. Eyes were washed in Tris-buffered saline (TBS) and dissected to remove conjunctiva, cornea, iris, ciliary body, lens, and retina leaving a posterior eye cup of RPE, choroid, and sclera. Eye cups were blocked in TBS + 5% Donkey serum (S30, Sigma-Aldrich), then treated with an anti-ICAM-2 primary antibody (1:500, Table 1), and Alexa Fluor 488-conjugated anti-rat secondary antibody (Table 1). Pictures were captured on a Ti2 widefield microscope (Nikon, Melville, NY). Area was quantified using Fiji by a reviewer blinded to animal identification.