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. 2020 Feb 19;6(8):eaax4568. doi: 10.1126/sciadv.aax4568

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Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 1 — (A) The distribution of TAP-tagged Na_V1.7 in the dorsal horn was detected using immunofluorescence with anti-FLAG antibody (left). The cross sections of spinal cord (lumbar 5) were costained with markers of superficial dorsal horn such as substance P (lamina I), PAP (lamina II), and vGLUT1 (lamina III onwards) antibodies (middle). The right panels show the merged left to middle panels. The results show that TAP-tagged Na_V1.7 mainly expresses in laminae I and II and in the part of lamina III. Scale bars, 100 μm. (B) Subcellular localization of TAP-tagged Na_V1.7 in the dorsal horn of the spinal cord was identified with immuno-EM techniques. Electron micrographs showing immunolabeling for FLAG in the dorsal horn of the spinal cord were detected using the pre-embedding immunoperoxidase (top) and immunogold techniques (bottom). Using the pre-embedding immunoperoxidase method, peroxidase reaction end-product for FLAG in the TAP-tagged Na_V1.7 mice was detected filling dendrites (Den) and dendritic spines of spinal cord neurons, as well as at presynaptic sites filling axon terminals (at). In the WT mice (top right), no peroxidase reaction end-product for FLAG was detected in the spinal cord. Using the pre-embedding immunogold method, immunoparticles for FLAG present in the TAP-tagged Na_V1.7 were mainly detected at intracellular sites (arrowheads in red), as well as along the plasma membrane (arrowheads in green) in dendritic shafts (Den) of spinal cord neurons. In addition, immunoparticles for FLAG were observed along the extrasynaptic plasma membrane (arrowheads in blue) of axon terminals (at). In the WT mice (bottom right), a very low density of immunoparticles for FLAG, similar to background levels, was observed attached to mitochondria in the spinal cord. Scale bars, 500 nm. HRP, horseradish peroxidase. (C) The number of immunoparticles in the superficial dorsal horn of TAP-tagged Na_V1.7 mice was counted. Of 1253 immunoparticles, 768 particles were located at postsynaptic sites (61%) and 485 particles were located at presynaptic sites (39%). Along the 768 postsynaptic particles, 501 particles were located at intracellular sites (65%), and 267 particles were located along the plasma membrane (35%). Along the 485 presynaptic particles, 204 particles were located at intracellular sites (42%), and 281 particles were located along the plasma membrane (58%).