Protocol for single-molecule FISH in the developing mouse retinal vasculature

The developing vasculature of the post-natal mouse retina is a powerful model to discover mechanisms of vessel formation and to test modulators of neovascularization. We present a protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas enabling the detection of individual mRNAs in vascular endothelial cells. We describe procedures from initial retina preparation to smFISH and detection. Our approach offers simple steps to overcome challenges related to tissue permeabilization, mRNA and protein co-detection, and post-acquisition image processing.