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. Author manuscript; available in PMC: 2019 Aug 23.
Published in final edited form as: Cell Rep. 2019 Aug 13;28(7):1935–1947.e5. doi: 10.1016/j.celrep.2019.07.037

Figure 2. Light and Electron Microscope Detection of Transported Biotinylated Proteins in the Visual Pathway.

Figure 2.

(A) Diagram of a sagittal section through the rat brain, adapted from the Paxinos atlas, showing the optic tract (OPT) and LGN (green) in the central boxed region and primary and secondary visual cortex (V1 and V2, respectively).

(B and C) Distribution of biotinylated proteins in the optic tract and LGN, detected with fluorescent streptavidin in sagittal brain sections from animals after intravitreal injection of NHS-biotin (B) or biocytin (C). Images are montages of single optical sections assembled in Photomerge. The montage in (C) was mounted on a black background. Scale bar in (C): 1 mm, applies to (B) and (C).

(D) Immunolabeled biotinylated proteins in the ON after intravitreal injection of NHS-biotin (top) or saline (bottom). Scale bar: 200 μm.

(E) Transmission immuno-electron micrograph of a cross section through the optic tract labeled with streptavidin-horseradish peroxidase (HRP) and DAB/nickel. Biotinylated proteins are contained within the axoplasm of RGC axons. Scale bar: 1 μm.

(F) Immuno-electron micrograph of a transverse section through the optic nerve labeled with 1.4 nm streptavidin-nanogold particles plus gold enhancement showing biotinylated proteins in RGC axons at low (top) and higher (bottom) magnification. The larger gold particles seen with gold enhancement indicate sites of greater biotin signal; individual particles outside of axons are background labeling.

(G-I) Intravitreal NHS-biotin injections label the RGC projections in LGN. Scale bar: 500 nm (G).

Confocal single optical section of fluorescent streptavidin immunolabeled, retinogeniculate axons showing intensely labeled boutons. Scale bar: 10 μm.

(H and I) Transmission immuno-electron micrographs showing the distribution of axonally transported biotinylated proteins labeled with avidin-HRP plus DAB (H) or streptavidin-nanogold (I) in presynaptic profiles in the LGN. Scale bars: 1 μm.