Mouse Retinal Histology Cryosectioning and Staining

Retinal histology was examined in cross sections based on prior protocols [22 (link)]. After euthanasia, enucleated mouse eyes were embedded and frozen in Optimal Cutting Temperature compound (Sakura Finetek, Torrence, CA, USA, Cat#: 4583). Cross sections (12 µm) were cut using a cryostat (Leica Biosystems, Wetzlar, Germany) and placed on positively charged microscope slides (VWR, Radnor, PA, USA, Cat#: 16004-406). Sections were air-dried and briefly fixed in 4% paraformaldehyde for 15 min and stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA, Cat#: HHS32-1L) for 3 min. Sections were then rinsed in ddH₂O and developed in tap water for 5 min, followed by 10 dips in acid alcohol (0.5% hydrochloric acid in 70% ethanol) to remove excess stain. Sections were then stained with eosin Y solution (Sigma-Aldrich, St. Louis, MO, USA, Cat#: 1098441000) for 30 s and rinsed in ddH₂O. The slides were dehydrated in a graded series of ethanol (50%, 70%, 95%, and 100%) and incubated with xylenes for at least 10 min. Slides were coverslipped using Permount Mounting Medium (Fisher Scientific, Waltham, MA, USA, Cat#: SP15-100) and air-dried before imaging.