Figure 6.

Detection of Wld^S and variant ΔNLS Wld^S proteins in nerves in vivo. A, Western blot showing presence of variant Wld^S proteins in sciatic nerve extracts from both spontaneous Wld^S mice and ΔNLS Wld^S transgenics. Note markedly higher levels of variant Wld^S protein in extracts from ΔNLS Wld^S lines 3 and 8 compared with Wld^S (heterozygous and homozygous). B, Western blot showing presence of Wld^S protein in sciatic nerve extracts from transgenic Wld^S rat (line 79, homozygous). For comparison, detection of Wld^S in similar total protein amount from brain homogenate from the same rat is also shown. C, Fluorescence immunostaining on wild-type, Wld^S (homozygous), and ΔNLS Wld^S (line 3, homozygous) longitudinal sciatic nerve sections using tubulin (green) and Wld18 (red) antibodies. Conventional epifluorescence microscopy (rows 1 and 2) demonstrates more intense Wld18 labeling in sciatic nerve from ΔNLS Wld^S mouse than from Wld^S. Higher-resolution confocal images (rows 3–6) from the same sections show variant Wld^S protein signals in glial cells whose nuclei were counterstained with Hoechst 33258 (blue). Note more prominent Wld18 staining in sample from ΔNLS Wld^S sciatic nerve. D, Confocal images showing induction of variant Wld^S expression (red) in glial cells 6 h after sciatic nerve injury located distally from the lesion site. Note nuclear Wld^S foci in activated glial cells (arrows, upper row) from native Wld^S mutant, whereas induced ΔNLS Wld^S protein expression shows a more cytoplasmic staining pattern demarcating the cell body (arrows, lower row). Blue, Hoechst 33258 counterstain. E, High-power confocal composite (z-series projection) shows presence and homogenous distribution of variant Wld^S protein (red) in the nodal and internodal axoplasm of ΔNLS Wld^S sciatic nerve (line 3) which is counterstained with tubulin (green). Note the fine granular staining pattern of Wld18 antibody within the axoplasm. Variant Wld^S protein immunoreactivity is absent from wild-type sciatic axons. F, Confocal images (z-series projection + DIC merge) showing localization of Wld^S protein (red) in nodal and perinodal axoplasm of native Wld^S axon in contrast to wild-type fiber where Wld^S signal is absent (Wld18 antibody plus Alexa568-tyramide signal amplification).