Whole mount staining of transected sciatic nerve at 5 and 7 days after injury.

<p>White arrows mark the proximal (left) and distal (right) nerve stumps and the red arrow within the proximal part of the nerve shows the limit of antibody penetration within this part of the nerve. A and B: whole mount stain of nerve preparations using neurofilament (NF) and S100β (S100) antibodies at 5 days (A) and 7 (B) days after nerve transection. C and D: higher magnification pictures of neurofilament and S100β stain showing interaction of distal Schwann cells (indicated by yellow arrow) with axons.</p

Whole mount staining showing angiogenesis and cell proliferation following nerve transection.

<p>A and B: staining with the endothelial cell marker CD31 and neurofilament (NF) at 5 days (A) or 6 days (B) after nerve transection identifies new blood vessel formation at the proximal and distal stumps of the transected nerve (identified by white arrows). C: whole mount sciatic nerve staining with myelin basic protein (MBP) and Ki67 antibodies to show presence of myelinated Schwann cells in proximal stump (MBP) and cell proliferation (Ki67) at 7 days after transection injury. Nuclei are counterstained with Hoechst dye (Ho). White arrows mark the proximal (left) and distal (right) nerve stumps.</p

Whole mount staining of transected sciatic nerve preparation after 90 days injury showing misdirected regrowing axons.

<p>A: phase image showing the structure of transected sciatic nerve, black arrows mark the proximal (left) and distal (right) nerve stumps. B: neurofilament (NF) antibody whole mount staining show regenerating axons in the nerve bridge and misdirected regrowing axons in both proximal and distal nerve stumps, blue arrows indicate that axons are growing outside of both proximal and distal nerve stumps. White arrows mark the proximal (left) and distal (right) nerve stumps. C: merged image of panels A and B.</p

Partnership of Schwann cells and axons during regeneration.

<p>Neurofilament (NF; green), S100β (S100; red) and Hoechst (blue) labelling of axons and Schwann cells at times shown after sciatic nerve transection. A-E: the front edges of the regenerating axons are covered by Schwann cell processes at 4, 5 and 7 days (d). At days 4 and 5, Schwann cell processes (red) form a ‘ball-like’ structure at the tip of axon bundles (A, B and E) whereas at 7d (C and D), fine Schwann cell processes appear to proceed in front of the axons. D: higher magnification of boxed area shown in panel C to show Schwann cell leading processes proceeding in front of axons and guiding axons across the nerve bridge. E and F: higher magnification from the boxed area of panel B. G: higher magnification of boxed area in panel F showing the axonal bundles, white arrows in panel G indicate apparent individual axons. Red arrows in D and H show elongated Schwann cell bodies held by axons crossing the nerve bridge. Upon further axon growth in panel H at 10d, elongated Schwann cell bodies can clearly be seen held by axons in the nerve bridge. In all the panels, the proximal side is up and distal to the bottom of the picture.</p

Re-growing axons enter the tibial nerve before other distal branches of the sciatic nerve.

<p>The sciatic nerve is stained with neurofilament antibody 5 days after nerve crush injury. At this timepoint, regenerating axons have grown much further within the tibial branch of the distal nerve (indicated by the white arrow) as compared to peroneal and sural branches.</p

Identifying the nerve injury site and leading regrowing axons after nerve crush injury using whole mount staining.

<p>Whole mount stained mouse sciatic nerve preparations at 5 days after crush injury. A-H: visualisation of crush site (indicated by red or white asterisk) by staining with neurofilament heavy chain (NF) antibody (A and E), Hoechst (Ho) dye (C and G), phase contrast (B) or Fluoromyelin, FM (F). Merged overlay panels in D (A-C) and H (E-G). I-L: neurofilament (I and J) and Hoechst (K) whole mount stain of nerve preparations distal to the crush injury showing the leading regenerating axons, indicated by white arrow, in J, and at higher power in panel I. L: merged overlay of panels J and K. In panels A-H, the proximal side is to the left and distal side to the right. In I, the proximal side is up and distal side to the bottom of the panel.</p

Double staining of Slit1-3 and Robo1-2 with neurofilament heavy chain in DRG.

<p>The neurofilament heavy chain antibody (NF) labels large diameter sensory neurons within the DRG. Merged images show that Slit1 (A), Slit2 (B), Slit3 (C), Robo1 (D) and Robo2 (E) are seemingly all expressed by large diameter sensory neurons. The yellow colour in merged images shows the co-localization of Slit1-3 and Robo1-2 signal with neurofilament heavy chain staining. Slit1-3 and Robo1-2 also appear to be expressed in small diameter cells in addition to their expression in large diameter sensory neurons. Slit1 (A) and Robo2 (E) show clear neuronal cell body staining. In contrast, Slit2 (B), Slit3 (C) and Robo1 (D) also show positive staining in the gaps between the cell bodies of sensory neurons in addition to their expression in the neuronal cell bodies.</p

Slit1-3 and Robo1-2 expression in the dorsal region of adult mouse spinal cord.

<p>Slit1-3 and Robo1-2 double staining with NeuN to show Slit1-3 and Robo1-2 expression in the dorsal region of adult mouse spinal cord. Spinal cord sections are from the L4/L5 region of adult C57BL/6 mice. Double staining with NeuN showed that Slit1 (A) and Robo1 (D) are not only expressed in dorsal neuronal cell bodies but are also expressed in other cells in the grey matter of the dorsal spinal cord. Slit2 (B) expression is low in dorsal spinal cord and seemingly restricted to neuronal cell bodies. Slit3 (C) and Robo2 (E) are also mainly expressed in the neuronal cell bodies in the dorsal spinal cord. Staining with Hoechst dye (Ho) is also shown (blue) to identify cell nuclei within the tissue.</p

Slit1-3 and Robo1-2 expression in the ventral region of adult mouse spinal cord.

<p>Images show Slit1-3 and Robo1-2 expression in the neuronal cell bodies localizing in the ventral horn of the spinal cord. (A) Slit1, (B) Slit2, (C) Slit3, (D) Robo1 and (E) Robo2. Slit1-3 and Robo2 are mainly expressed in the neuronal cell bodies in the ventral horn of the spinal cord. Robo1 is highly expressed in the ventral horn neuronal cell bodies of the spinal cord but Robo1 also appears to show expression in other cells in the grey matter of the spinal cord (D). Staining with Hoechst dye (Ho) is also shown (blue) to identify cell nuclei within the tissue.</p

Slit2, Slit3 and Robo1 expression in the cell bodies of non-myelinating Schwann cells.

<p>Higher magnification images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172736#pone.0172736.g013" target="_blank">Fig 13</a> show the expression of Slit2, Slit3 and Robo1 in non-myelinating Schwann cells. Non-myelinating Schwann cells in the PLP-GFP mouse nerve could be easily distinguished by the morphology of Remak bundles via the GFP signal (A-C). Slit2 (A), Slit3 (B) and Robo1 (C) expression in non-myelinating Schwann cells was observed (indicated by white arrows). Slit2 and Robo1 staining appeared stronger in the small diameter axons of the Remak bundle rather than the non-myelinating Schwann cells. Slit3 showed strong expression in non-myelinating Schwann cells.</p