Figure 1

Antibodies accumulate in peripheral nerves of myelin mutant mice. (A) Immunohistochemical localization of endogenous antibodies (IgG) in the femoral quadriceps (q) and saphenous nerve (s) from 6-month-old wildtype mice (P0wt) and myelin mutants (P0het). Note the prominent labeling of the endoneurium in nerve cross sections in femoral quadriceps nerves of mutants in contrast to wildtype mice. RAG1-deficient mice (deficient for T- and B-lymphocytes) are, expectedly, devoid of antibody deposition. Scale bar, 20 μm. (B) IgG fluorescence intensity in the endoneurium of femoral quadriceps nerve cross sections is shown as the mean gray value, taking P0wt nerves as a reference. n = 2 animals per genotype were analyzed within three repeated stainings and measurements. (C) Immunohistochemical localization of endogenous antibodies (IgG) in the femoral quadriceps (q) and saphenous nerve (s) from 1-month-old wildtype mice (P0wt) and myelin mutants (P0het). Note similar endoneurial immunoreactivity in comparison to 6-month-old mutants (see A), but the lower intensity of the perineurium. Scale bar, 20 μm. (D) Immunocytochemical staining against IgG on single fiber preparations. Note preferential deposition of antibodies in the nodal regions and the generally stronger signal along myelinated fibers in P0het mice compared to wildtype mice. Nerve-specific antibodies are not detectable in RAG1-deficient mice. The corresponding phase contrast micrograph displays the teased fiber with a node of Ranvier (arrowheads). Scale bar, 20 μm. (E) Immunocytochemical staining against IgG or β-III-Tubulin on unfixed and non-permeabilized native sciatic nerve fibers of P0het mice (left and middle column). Note IgG immunoreactivity along the outer margins of the myelinated nerve fibers and at nodes of Ranvier (arrowhead, left column) whereas β-III-Tubulin-immunoreactivity is confined to some axonal profiles at nodes of Ranvier (arrowheads, middle column), likely due to the vulnerability of this structure upon teasing. In the permeabilized sciatic nerve samples of P0het mice, β-III-Tubulin immunoreactivity is now visible along the entire axon (arrows, lower right column) and no longer confined to nodes (see middle columns), whereas IgG immunoreactivity is still detectable along myelinated fibers (upper right column). White dashed lines indicate outer margins of myelinated nerve fibers. Scale bar, 20 μm. (F) Western blot analysis of wildtype (P0wt) and P0het femoral quadriceps nerve lysates showing stronger IgG signal in mutant mice. RAG1-deficient mice show no antibody signal. ERK1/2 serves as a loading control. (G) Densitometric analysis of IgG immunoreactivity in Western blots from P0het mutant mice and P0wt littermates, taking ERK1/2 signal as a reference (loading control). Results of n = 2 to 3 from two independent experiments are depicted.