After enucleating the eyes, the anterior segments were removed and the retina was carefully excised. Eyecups were then fixed overnight with 2% PFA at 4°C. After washing, four pie cuts were made to allow flattening of the choroid/sclera eyecup as well as clearly isolate superior and inferior quadrants. Then the choroid was prepared for IHC as previously described (19 (link)). The choroid was blocked with 2% normal goat or donkey serum for 4 h at 4°C, washed in 0.1% triton X-100 in TBS, and then incubated with a mixture of the following primary Abs: mouse anti-RPE65 (1:200, NB100–355, Novus Biologicals, Centennial, CO, USA), goat anti-Iba1 (1:200, ab5076, Abcam, Cambridge, MA, USA) and rabbit anti-MC tryptase (1:200, CAU26568, Biomatik, Wilmington, DE, USA) overnight at 4°C. After washing, they were incubated with Alexa-Fluor 488 or Cy3 conjugated goat or donkey secondary Abs (1:300, Jackson ImmunoResearch, West Grove, PA, USA) overnight, followed by mouse anti-ZO-1 Ab conjugated with Alexa-Fluor 594 (1:100, 339194, Thermo Fisher Scientific, Waltham, MA, USA).