Cd3 clone f7.2.38

Antibodies against CD3 (clone F7.2.38; DAKO, Glostrup, Denmark) for T cells and CD68 (clone ED1; Bio-Rad, Hercules, CA, USA) for macrophages were used for immunohistochemistry. Deparaffinized sections were pretreated with microwave in Tris-EDTA buffer (pH 9.0) and with proteinase K (DAKO) (100 μg/mL, 37 °C, 10 min) for CD3 and CD68, respectively. Sections were then incubated with 5% skim milk in phosphate buffered saline (PBS) for 15 min and with primary antibody for 1 h, followed by 1 h incubation with peroxidase-conjugated secondary antibody (Histofine simple stain MAX PO; Nichirei Biosciences, Tokyo, Japan). Positive reactions were visualized with 3,3′-diaminobenzidine (DAB; Nichirei Biosciences; Tokyo, Japan). Sections were lightly counterstained with hematoxylin. In order to evaluate hepatocellular apoptosis, liver sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method as previously described [19 (link),20 (link)]. The number of TUNEL-positive apoptotic hepatocytes was counted; more than 500 hepatocytes were analyzed in each animal to obtain reliably quantitative data.

Minor LSG biopsy samples were obtained at W0 and W28. Samples of LSGs were fixed in alcohol, acetic acid and formaldehyde solution and embedded in paraffin wax for histological study. All samples were analysed by the same pathologist (TL).
The following parameters were analysed: the focus score, the B-cell/T-cell ratio in the foci, BAFF expression in the foci and the NK infiltrate inside and outside the foci. The focus score was measured after haematoxylin eosin staining and was expressed as the number of foci (50 lymphocytes) per 4 mm². The focus score was considered abnormal if it was ≥1. B cells and T cells were identified after CD20 (clone L26; DakoCytomation, Glostrup, Denmark) and CD3 (clone F.7.2.38; DakoCytomation) staining, respectively. The B-cell/T-cell ratio was estimated in foci and expressed as the proportion of B cells to T cells. BAFF expression was measured after incubation with a rat anti-human BAFF antibody called Buffy-2 (Enzo Life Sciences, Villeurbanne, France) and expressed as the proportion of BAFF-expressing cells inside the foci, regardless to their origin (B or T cells). NK cell infiltration was analysed after incubation with mouse anti-human NKP46/NCR1 antibody (R&D Systems) inside and around the foci and was expressed as the number of NK cells per square millimetre as previously described [17 ].

Stainings for calprotectin-expressing phagocytes (clone MAC387, Bio-Rad, CA, USA) or T lymphocytes (CD3, clone F7.2.38, Dako, Denmark) were performed as described previously^{10 (link)}. The number of positive cells was counted in one cross-section from each construct and averaged per group.
To stain blood vessels, samples were incubated with anti-CD31 (clone JC70A, 1:50, Dako) for 1 h at room temperature following heat-induced antigen retrieval with EDTA (1 mM, pH 9.0). Blocking and staining was performed with a kit, according to the manufacturer’s protocol (Dako EnVision + System HRP, Dako).
Using the same kit as the CD31 staining, cytoplasmic staining for connective tissue macrophages (clone RAM11, 1:100, Dako) was performed with overnight incubation of the primary antibody at 4 °C. Sections were counterstained with Mayer’s hematoxylin.

Sural nerve biopsy was performed after obtaining informed consent, as previously described. Light and electron microscopy preparations, as well as teased fiber analysis, were performed according to standard methods [20 (link)]. Immunohistochemistry was performed in selected cases (see below). Sections were deparaffinized in xylene, rehydrated through decreasing concentrations of ethyl alcohol and then rinsed in distilled water. Antigen retrieval was performed in a 90 °C solution of 0.001 M EDTA buffer (pH 8.0) for 20 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in distilled water. Slides were incubated with antibodies recognizing CD3 (clone F7.2.38, 1:50, Dako), CD20 (clone L26, 1:50, Dako) and CD68 (clone KP1, 1:1000, Dako) for 1 h at room temperature. Bound antibodies were detected using Envision FLEX/HRP and 3–30-diaminobenzidine as chromogen (Dako). Slides were counterstained with haematoxylin (Sigma-Aldrich), dehydrated and mounted.

Immunolabeling for flaviviral NS1 antigen has been previously described in detail [9 (link)]. Immunophenotyping of rabbit T lymphocytes and histiocytes in inflammatory aggregates was achieved using cross-reactive anti-human CD3 (CD3 clone F7.2.38, Dako) and anti-human myeloid/histiocytic antigen (Clone MAC387, Dako) mouse monoclonal antibodies, respectively, using previously described protocols [60 (link)]. For each IHC batch, a positive and negative antigen control was included.