Immunohistochemical Analysis of Mouse Eyes

Age-matched mice were euthanized, and enucleated eyes were fixed for 24 h in 1:10 buffered formalin. Fixed eyes were paraffin embedded, and thin sections were immunostained as described (Asthana et al., 2017 (link)). In brief, sections were rehydrated with xylene followed by decreasing concentrations of ethanol (100%, 90% and 70%), and finally, three washes of 5 min each with 1× PBS. Antigen retrieval was performed by heating the tissue at 97 °C in the presence of 25 mM tris-1 mM EDTA (pH-8.5) for 30 min. Non-specific sites were blocked with 1% BSA for mouse and human tissue, and NAP blocker (G-biosciences, St. Louis MO) for bovine tissue. The tissues were subsequently washed and incubated with respective primary antibody followed by incubation with the corresponding Alexa Fluor conjugated secondary antibodies. The nuclei were stained with Hoechst (#33342, Invitrogen, USA) and the sections were mounted using Fluoromount-G (Southern Biotech, USA). Images were captured with Leica inverted microscope (DMi8).