Figure 5

Figure 5

Inhibitory effect of 3f on the phenotypes of PND. (A) Immunostaining was performed in explant sciatic nerve fibers. Neurofilament (NF, green) and 4′,6-Diamidine-2′-phenylindole (DAPI, blue) were used as markers for axons and nucleus, respectively. NF index was calculated by counting the 100 µm-long consecutive lines (n = 3 mice). Scale bar, 50 µm. 3DIV, 3 days in vitro. Con, control. (B) Myelin sheath was identified using an anti-myelin basic protein (MBP, red) antibody counterstained with DAPI. Scale bar, 50 µm. MBP index was estimated by calculating the 100 µm-long consecutive lines of MBP-marked nerve fibers (n = 3 mice). (C) Immunostaining with lysosomal-associated membrane protein 1 (LAMP1, green) combined with DAPI (blue) in ex vivo sciatic nerve fibers showed the inhibitory of the 3f on Schwann cell dedifferentiation. Scale bar, 50 µm. The intensity of LAMP1 expression was quantitatively measured in the teased sciatic nerve fibers within 200 × 200 μm² widths of teasing slides (n = 3 mice). The intensity at 3DIV was set as 100%. (D) Cyclin D1 (CCND1, red) and DAPI (blue) were immunostained in ex vivo sciatic nerve fibers. The arrows indicated CCND1/DAPI double-positive cells. Scale bar, 50 µm. Quantification was proceeded by calculating CCND1-marked cells out of 200 DAPI-positive cells and then compared to that of the nerve fibers at 3DIV (n = 3 mice). * p < 0.05, *** p < 0.001.