Surviving neurons continue to express NF180 protein after axotomy.

<p>In an animal 10 months post-TX at the level of the 5<sup>th</sup> gill, DTMR was applied to a second complete TX at the level of the 3rd gill (approximately 5 mm below the obex of brain and 2 mm rostral to the original TX site), in order to label neurons projecting to the spinal cord, regardless of whether their axons had regenerated. After 5 days to allow for retrograde labeling, the brain was removed, pinned flat, and photographed live before fixation. <b>A,</b> fluorescently-labeled RNs superimposed on a brightfield image of the living brain. <b>B,</b> After fixation, the same brain was stained for NF180. DTMR labeling correlated closely with NF180 immunostaining, but there were exceptions: 1) Two swollen neurons (* in <b>A</b>; probably the left B1 and B4) were labeled by anti-NF180 but were not backfilled by DTMR, indicating that they had survived but their axons had retracted long distances and had not regenerated back to the level of the 3<sup>rd</sup> gill. 2) Some neurons were labeled by DTMR but not by NF180. Prominent among these were neurons in the medial inferior reticulospinal group (m.i.r.), reflecting that not all spinal-projecting neurons express NF180 in animals of this age [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137670#pone.0137670.ref017" target="_blank">17</a>]. The faint stain just caudal to B1 (white arrowhead in <b>A)</b> is a small neuron, not collapsed B1 cytoplasm, as shown in the inset of <b>B</b>. The white arrows in <b>A</b> and <b>B</b> point to neurons that are double-labeled because they have axons that regenerated and also express NF180.</p>