biopsies as CIDP or CIAP in about 70% of the cases. It is important to note that these observers were not trained, and the inter-observer variability suggests that the training of the observers may improve the accuracy. Future studies will examine whether they can be applied to less invasive biopsy procedures such as muscle or skin biopsies. In summary, our study characterizes for the first time the profound alterations of nodes of Ranvier in human chronic acquired neuropathies using both electron microscopy and immunofluorescence, and shows that alterations in paranodin immunofluorescence may provide an interesting contribution to the diagnosis. Materials and Methods Patients The present study was carried out using remaining material of biopsies previously carried out for diagnostic purposes. Patients diagnosed with CIDP and CIAP are described in Tables S1 and S2. Biopsies were carried out solely for diagnosis purposes before the present study was initiated and thus, did not need the approval from the ethics committee. Patients gave their written consent at the time of biopsy, for further use of possible remaining material for research purposes. Control nerves were a superficial peroneal nerve and terminal motor branches of intramuscular nerves in muscle biopsies devoid of lesion. Biological investigation included complete blood cell count, sedimentation rate, fasting glucose level, creatininemia, thyroid function, liver function, vitamin B12, folic acid, assay for anti-nuclear antibodies, complement, serologies for human immu- nodeficiency virus, B and C hepatitis, Lyme disease, search for cryoglobulinemia, monoclonal gammapathy, and cancer. Diagnosis of CIAP was made in patients with: i) Purely distal polyneuropathy, involving sensory (patients 19 and 21) or motor and sensory modalities (patients 13–18, 20, 22); ii) Electrophysiological findings of axonal degeneration; iii) Absence of abnormalities on biological investigation; iv) Slowly progressive course after a two-year follow up (patients 15 and 17 had a nerve biopsy less than two years after the beginning of the disease but further follow-up did not change the diagnosis); v) Absence of the Ad Hoc Subcommittee of the American Academy of Neurology (AAN) criteria for nerve demyelination and absence of other identifiable cause of polyneu- ropathy (absence of vasculitis, granuloma or abnormal deposits) [28]. Diagnosis of CIDP was based on typical clinical and ENMG findings in patient 1 only, and in the others on: i) Very suggestive clinical presentation with proximal limbs involvement (patients 5, 6), pronounced ataxia (patients 3, 4, 5), diffuse areflexia (patients 5, 8, 11), relapsing course (patient 6), involvement of cranial nerves (patients 7, 8) and ii) presence of some ENMG criteria for demyelination (patients 2, 7, 9). These suggestive combination of clinical and electrophysiological criteria led to measure the CSF protein content, which was abnormally high in patients 2, 4, 6, 7, 9, and 12, and to perform a nerve biopsy, which disclosed the AAN Ad Hoc Subcommittee histological criteria for demyelination [28] in patients 2, 4, 6, 8, and 10–12. Fifty eight percent of the patients (patients 1–3, 5, 7, 8, and 10) responded favorably to immuno- modulating treatment. Mice Mice with conditional Krox20 inactivation were produced as described [27]. In brief, these mice carried one allele in which Krox20 coding sequence was replaced by beta-galactosidase sequence (lacZ) and one allele in which LoxP sequences allowed the excision of a critical fragment of the Krox20 coding sequence following the action of Cre (floxed gene). These mice also carried a transgene, R26-CreERT, coding for Cre fused to a mutated form of Acquired Neuropathies the estrogen receptor (ERT) able to bind tamoxifen but not endogenous estrogens, which prevents the nuclear activity of Cre. For inducing Krox20 inactivation, Krox20lacZ/flox, R26-CreERT mice were injected with tamoxifen (10 mg/ml, i.p.), in a 10:1 sunflower oil/ethanol solution, 100 ml twice a day for 5 days. Control mice received only vehicle. Mice were killed and nerves prepared 28 days after this treatment. The total number of mice used was six and all experiments were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87849, license B75-05-22). Our Institute has the authorization for the use of mice without approval of specific research project. Biopsy nerve preparation and electron microscopy Superficial peroneal nerves were obtained from patients for diagnosis purposes, previously to the present study, under local anesthesia, at the lower third lateral part of the leg. One centimeter was fixed in 2.5% (weight/vol) glutaraldehyde in sodium phosphate buffer (pH 7.4, 150 mM), post-fixed in 2% (weight/vol) osmium tetroxide in the same buffer and embedded in Epon. Semi-thin sections (0.5 mm) were stained with toluidine blue and examined under light microscope. One centimeter was orientated longitudinally, mounted on a cork disk with tragacanth gum and frozen in isopentane cooled by liquid nitrogen at 2160uC. Electron microscopy was carried out on 2 samples from patients with CIAP and 2 with CIDP at the Institut du Fer a` Moulin Cell and Tissue Imaging Facility with a Philips CM 100 microscope equipped with a Gatan Orius 832 camera for digitized acquisition. Nodal regions were located at low magnification and extensively studied at higher magnification. Antibodies Rabbit antibodies against paranodin (L51) have been described [17]. Voltage-gated Na+ channel a subunit mouse monoclonal antibody (PAN Nav, clone K58/35) and rabbit polyclonal anti- sodium channel (PAN-SP19) were from Sigma-Aldrich (St Louis, MO). Rabbit antibodies for KCQN2 [15] were a gift of Je´roˆ me Devaux (CNRS, Marseille, France). Rabbit antibodies for ankyrin G [38] and neuronal class III b-tubulin (TUJ1), and mouse monoclonal antibody for ankyrin G were from Chemicon Inc. (Temecula, CA). Tissues extracts and immunoblotting Mouse sciatic nerves were dissected out and frozen in isopentane pre-cooled with liquid nitrogen. Samples were stored at 280uC. Tissue fragments (,1 mm3) from frozen nerves were homogenized in a buffer containing 10 mM sodium phosphate, pH 7.8, 60 mM NaCl, 1% (vol/vol) Triton X-100, 0.5% (weight/vol) deoxycholic acid, 0.1% (weight/vol) SDS, 10% (vol/vol) glycerol, 25 mM alpha- glycerol-phosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and Complete protease inhibitor (Roche Applied Science). Insoluble material was removed by centrifugation at 21,0006g for 10 min at 4uC. Protein concentration was determined using microBCA (Pierce). Extracts were in NuPAGE LDS were resolved on 4–12% NuPAGE BisTris gels (Invitrogen), and transferred to nitrocellulose mem- branes. Membranes were blocked and incubated with antibodies as described [39]. Primary antibodies were detected using IRDye 700- or 800-conjugated secondary antibodies (Rockland) and Odyssey Imaging System (LI-COR biosciences). Immunofluorescence Longitudinal sections of frozen human superficial peroneal nerve biopsies or mouse sciatic nerves were cryostat-sectioned (8– PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e14533