E0413

The following antibodies were used: Polyclonal rabbit antibody against human myeloperoxidase (MPO) (A0398, Dakocytomation, Glostrup, Denmark); polyclonal rabbit anti-muc2c3 (kindly provided by the University of Gothenburg, Gothenburg, Sweden); polyclonal rabbit anti-SAM-pointed domain containing ETS transcription factor (SPDEF) (ab197375, abcam, Cambridge, United Kingdom); monoclonal rabbit anti-BiP (C50B12, cell signaling technology, Leiden, The Netherlands); monoclonal mouse anti-CHOP (clone 9C8, MA1-250, Thermo Fisher Scientific, Waltham, MA, USA), and polyclonal rabbit anti-cleaved caspase-3 (CC3) (asp175) (9661, cell signaling technology, Leiden, The Netherlands).
Secondary antibodies used were: Peroxidase-conjugated goat anti-rabbit (111-035-045, Jackson, West Grove, PA, USA), biotin-conjugated rabbit anti-mouse (E0413, DakoCytomation, Glostrup, Denmark), and biotin-conjugated swine anti-rabbit (E0353, DakoCytomation, Glostrup, Denmark).

For cyclin A immunohistochemistry, FFPE tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was done by heating in Tris buffer and endogenous peroxidase activity was blocked by incubating the slides in a solution of 0.3% hydrogen peroxide in phosphate-buffered saline. Primary antibody (Leica, Novocastra, Newcastle upon Tyne, United Kingdom: monoclonal, mouse) with a dilution of 1:200 was incubated overnight at 4°C. Rabbit antimouse (1:150; E0413, Dako, Heverlee, Belgium) was used as secondary antibody. Visualization was achieved by using the horseradish peroxidase avidin–biotin complex (HRP-ABC) method and diaminobenzidine (DAB) substrate. Finally, slides were counterstained with hematoxylin. A negative control was obtained by omission of the primary antibody. Positive nuclei in the proliferation zone of the BE epithelium were used as internal positive control. Immunohistochemical staining for p53, AMACR, and SOX2 was performed as previously described.^{[16 (link)–18 (link)]}

96-well ELISA plates were coated overnight at 4°C with 1 μg/ml of T-AChR diluted in 10 mM NaHCO3 buffer, pH 9.6. T-AChR coated wells were blocked with 10% SVF in PBS at 37°C for 2-3 hours. 100 μl of mouse serum (1/100,000) per well were incubated for 90 min at 37°C. Subsequently, wells were washed 4 times with the PBS-Tween buffer. 100 μl of 1/10,000 diluted biotinylated anti-mouse IgGs (E0413, Dako, Courtaboeuf, France) were added for 90 min at 37°C. Next, samples were incubated with 100 μl of streptavidin-horseradish peroxidase 1/20,000 (PN IM0309, Beckman Coulter, Villepinte, France) for 30 minutes and tetramethylbenzedine was used for color development. To determine the optical density (OD) at 450 nm, we used a microtiter plate reader spectrophotometer.

To detect anti-AChR antibodies, 96-well ELISA plates were coated overnight at 4°C with 0.5 µg/mL of T-AChR diluted in 10 mM NaHCO3 buffer, pH 9.6. T-AChR-coated wells were blocked with 10% SVF in PBS at 37°C for 2–3 h. 100 µL of mouse serum (1:100,000 for EAMG experiments or 1/100 for other experiments) or 5 µg of thymic extracts were used per well and incubated for 90 min at 37°C. Subsequently, wells were washed four times with a PBS-0.05% Tween buffer. 100 µL of 1/10,000 diluted biotinylated polyclonal anti-mouse IgGs (E0413, Dako, Courtaboeuf, France) were added for 90 min at 37°C. Next, samples were incubated with 100 µL of streptavidin–horseradish peroxidase 1:10,000 (PN IM0309, Beckman Coulter, Villepinte, France).
To measure the expression level of all IgG, 96-well plates were coated overnight at 4°C with a polyclonal goat anti-mouse IgGs (1 µg/mL) (Z0420, Dako). After blocking for 1 h with PBS-BSA 2%, mouse serum (1:100,000) or standards (Clone DAK-GO1, X0931, Dako) were incubated for 90 min at 37°C and, subsequently, 1:10,000 diluted biotinylated anti-mouse IgGs and 1:10,000 streptavidin–horseradish peroxidase were added.
Tetramethylbenzidine was used for color development, and the optical density at 450 nm was measured with a microtiter plate reader spectrophotometer. The levels of anti-AChR antibodies were normalized on the total level of IgG.

3-NT staining was performed as described by Haesen et al. [4 (link)]. In short, heat-mediated antigen retrieval was performed in deparaffinised 8 µm aortic tissue sections using citrate buffer (pH 6), and sections were washed with PBS and used for 3,3′-diaminobenzidine immunostaining. The sections were incubated with a primary antibody against 3-NT (1:100, mouse monoclonal, Ab7048, Abcam) and were diluted in PBS for 1 h at room temperature. A biotinylated anti-mouse antibody (1:100, E0413, Dako) and streptavidin-HRP (1:400, P0397, Dako) were both applied for 30 min at room temperature. After immunostaining, the nuclei were counterstained using haematoxylin and embedded in a DPX mounting medium. Images were acquired using a Leica MC170 camera connected to a Leica DM2000 LED microscope. The level of staining was assessed in six random fields per section using the colour deconvolution plugin in ImageJ software [61 (link)] and was expressed as the % of the total surface area of interest. Two operators blinded for group allocation performed the analysis independently.

ELISA experiments were carried out on serum samples collected at sacrifice (day 43). 96-well plates were coated with 0.5 μg/mL of T-AChR diluted in 10 mM NaHCO3 buffer, pH 9.6, overnight at 4 °C. Wells were blocked with PBS 10% fetal calf serum for 2h30 at 37°C. Samples were diluted in PBS 0.2% BSA (1:100 000) and incubated at 37 °C for 1 h and 30 min. One hundred microliters of biotinylated rabbit anti-mouse IgG (1/10000, E0413, Dako, Courtaboeuf, France) or biotinylated anti-mouse IgG subtypes were added for 1 h and 30 min at 37 °C (anti-IgG2b 1/250 (553393, BD Biosciences, Le Pont de Claix, France); anti-IgG2c (1/5000, SA5-10235, ThermoFisher Scientific) or anti IgG1 (1/100; 553441, BD Biosciences). Samples were incubated 30 min with 100 μL of streptavidin–horseradish peroxidase (diluted at 1:10 000) (S911, ThermoFisher Scientific). Tetramethylbenzidine was used for color development, and the optical density at 450 nm was measured with the SPARK 10 M microplate reader (TECAN Life Sciences, Grödig, Austria). Between each step, wells were washed 4 times with 200 μl of PBS 0.05% Tween 20.