Figure 4.

Macrophages in the Outermost Layer of the Nerve Bridge Express Slit3

(A–C) Time course of Slit2 (A), Slit3 (B), and Robo1 (C) mRNA fold changes in the nerve bridge following injury, n = 3. ^∗p < 0.05, ^∗∗p < 0.01 compared with the intact nerve.

(D) Slit3 and neurofilament (NF) double staining on nerve bridge transverse section showing that Slit3-positive cells form the outermost layer of the nerve bridge.

(E) F4/80 and NF double staining showing that the outermost layer of the nerve bridge is formed by a high density of macrophages.

(F and G) Slit3 and F4/80 double staining on a transverse (F) and longitudinal (G) nerve bridge sections confirming that macrophages in the outermost layer of the nerve bridge express Slit3.

(H–J) Higher magnification images from (G) showing that macrophages in the outermost layer of the nerve bridge express high levels of Slit3 (H), macrophages in the middle of the nerve bridge also express Slit3 (I), but macrophages in the distal nerve stump express low levels of Slit3 (J).

Blue signal in (D)–(J) represents cell nuclei staining (Hoechst). Scale bar in (D)–(G) represents 100 μm and in (H)–(J) represents 10 μm.

Several images were captured on a Zeiss LSM510 confocal microscope in (D) and (E) and combined into one image using Adobe Photoshop software (Adobe Systems). Several z series were captured on Zeiss LSM510 confocal microscope in (G), covering the entire field of interest. The individual series were then flattened into a single image for each location and combined into one image using Adobe Photoshop software (Adobe Systems).