Using methanol to preserve retinas for immunostaining

Experimental studies on retinal vasculature and retinal ganglion cells (RGCs) investigating the developmental and pathological conditions of the retina mainly rely on whole-mount retinal immunostaining. Methanol, an auxiliary fixed medium for retinal whole-mount preparations, has been used in some studies; however, its application in short- and long-term storage of retinas for further study has not been well described. We aimed to evaluate methanol use as a preservation treatment for further immunostaining of the retina.

We generated oxygen-induced retinopathy (OIR) and optic nerve crush (ONC) mouse models and used their retinas for analysis. We pipetted cold methanol (-20°C) on the surface of the retina to help fix the tissues while promoting permeability, after which the retinas were stored in cold methanol (-20°C) for 1, 6, or 12 months before being evaluated using various optical techniques. Thereafter, retinal whole-mount immunostaining was performed to analyse retinal neovascularisation and retinal hypoxia in OIR model, and retinal ganglion cell survival rate in ONC model.

Quantitative analysis revealed no significant differences in the fixed retinas after long-term storage in terms of retinal vasculature or retinal hypoxia in the OIR model. Similarly, no significant difference was found in RGC survival rate after long-term storage in methanol. These results suggest that methanol can be used as a storage medium when preserving retinal whole-mount samples.

Cold (-20°C) methanol can serve as an effective medium for long-term storage of fixed retinas, which is useful for further research.