Hep 20 10 slides

Ready to use HEp-20-10 slides (Euroimmun; product No. FA 1512-20) were stained according to manufacturer’s instructions with minor modifications. Recombinant mAbs (mouse IgG1) were diluted to 5 µg/ml or 25 µg/ml in block buffer (0.1% tween, 0.5% BSA in PBS). After one hour of incubation with the mAbs, slides were washed in block buffer and stained with goat anti-mouse IgG-Alexa488 (Life Technologies) and 2 µg/ml DAPI (Sigma) for one hour. After washing, coverslips were mounted with Fluoro-gel (Electron Microscopy Sciences). Images were acquired using an Olympus Fluoview FV1000 confocal system.

Samples were analyzed on HEp-20-10 slides in a 1/100 dilution (Euroimmun, Lübeck, Germany). The assay was conducted according to manufacturer’s instructions. The cut-off decision for positivity is an antibody titer of 1/100. If positive, samples were titrated (1/320 and 1/1000). Images of the incubated slides were automatically acquired by the EUROPattern microscope (Euroimmun, Germany) and digitally transferred into the software for further evaluation. Images were interpreted, once automatically by the EPa software (Euroimmun, Lübeck, Germany) and, in a parallel approach, visually by two independent individuals (Observer 1 and 2). IIF images were evaluated with respect to negativity/positivity, pattern (homogeneous, speckled, nucleolar, centromere, nuclear dot, nuclear membrane, cytoplasmic) and titer (1:100; 1:320; 1:1000; > 1:1000).