Permount mounting media

PAS (Periodic Acid-Schiff)/hematoxylin staining was used to detect the structure changes of retinal vessels.37 (link) The retinas were fixed in 4% paraformaldehyde, dissected out from the eyecups, and digested in 3% trypsin for 2–3 h at 37 °C. Retinal vessels were separated from other retinal neuronal cells by gentle shaking under a dissection microscope. The vessels were mounted on a slide, allowed to dry, and stained with PAS/hematoxylin (Glycogen PAS Stain Kit). After staining and washing in water, retinal vessels were dehydrated and mounted using the Permount mounting media (Sigma, St. Louis, MO, USA). Retinal vasculature was observed under a microscope (DP80, Olympus, Japan). Acellular capillaries were counted from the images for each retina and expressed as the number of acellular vessels per mm².

Retinal vasculature was prepared using trypsin digest as described previously.⁶³ (link) In brief, eyes were fixed in 4% paraformaldehyde freshly made in PBS overnight. Retinas were dissected out from the eyecups and digested in 3% trypsin (Gibco-BRL) for 2–3 h at 37°C. Retinal vessels were separated from other retinal neuronal cells by gentle shaking and manipulation under a dissection microscope. The vessels were then mounted on a clean slide, allowed to dry, and stained with PAS-H&E (periodic acid solution-hematoxylin, Gill No.3, Sigma, St. Louis, MO, USA) according to the instruction manual. After staining and washing in water, the tissue was dehydrated and mounted using Permount mounting media (Sigma, St. Louis, MO, USA). The prepared retinal vessels were imaged using the Leica LAS X widefield systems. At minimum, 10 representative, non-overlapping fields from each quadrant of the retina were imaged. Acellular capillaries are counted from images for each retina and expressed as number of acellular vessels per mm².