Intravitreal NHS-Biotin Injection and Immunohistochemistry to Label and Image Protein Transport in the Mouse Optic Nerve

The process of moving proteins and organelles along the axon is essential for neuronal survival and function, ensuring proper communication between the cell body and distant synapses. The efficient and precise delivery of proteins via axon transport is critical for processes ranging from synaptic plasticity and neurotransmission to neuronal growth and maintenance. However, the identities of all the transported proteins have only recently begun to be investigated. Retinal ganglion cells (RGCs) provide a unique opportunity for access to central nervous system (CNS) axons as the retina is located outside the brain in the eye, with long axonal projections (~1 cm in mouse) that innervate the brain. We have developed and optimized methods for unbiased in vivo protein labeling in rodent RGC somata with intravitreal N-hydroxysuccinimido (NHS)-biotin and subsequent visualization of transported proteins along the optic nerve using confocal microscopy. Here, we describe these procedures in detail. Key features • Expands on the intravitreal injection method presented in Schiapparelli et al. [1] and extends it to whole mount optic nerve immunolabeling. • Unbiased in vivo labeling of proteins in the mouse central nervous system. • Optimized mouse intravitreal injection procedure using glass needles to reduce injury and costs. • Immunofluorescent imaging of the cleared intact optic nerve to visualize transported labeled proteins.