Bx53 upright fluorescence microscope

Sera were obtained from whole blood through centrifugation at 2,000g for 15 min and then stored at −20°C until use. Antinuclear and cytoplasmic antibodies were tested by IIF on HEp-2 ANA slides (INOVA Diagnostics, San Diego, CA, USA) using serial dilution of human sera (1:80; 1:160; 1:320; 1:640; 1:1,280) of patients and healthy patients’ sera as control for autofluorescence followed by AlexaFluor488 AffiniPure F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch Europe Ltd., Suffolk, UK) as previously described (20 (link)). Images were acquired using the Olympus BX53 Upright fluorescence microscope.
After HEp-2 IIF analysis, we performed the IIF analysis on tissue slides for all the patients who had variable expression of autoAbs directed against the PDC, following the manufacturer’s instructions (Astra Formedic, Milan, Italy). The kit included also positive and negative controls, and protocol procedures are the same as described above for IIF on HEp-2 slides. IIF patterns were reported according to the ICAP nomenclature (https://www.anapatterns.org; Supplementary Table 1), and we used their reference sera (including AMA) as positive control for autoAb analysis (10 (link)).

Purified neutrophils (6 × 10⁵) in 200 μl conditioned medium (CM) were seeded into the upper chambers of transwell plate containing a polycarbonate filter (3 μm pores, Corning, UK). CM (500 μl) was added into the lower chambers in a 24-well trans-well plate. To detect neutrophil migration stimulated by IL-17A, 100 ng/ml IL-17A was added to the upper chambers. After 16 h, the translated neutrophils in the lower chambers were imaged using an IX73 inverted fluorescence microscope (Olympus Corporation, Japan). The translated neutrophils on the bottom of the upper chambers were fixed with precooled methanol at 4 °C and stained with crystal violet (Sigma, 548-62-9). After washing, the neutrophils at the bottom of the upper chambers were gently flicked, and the exterior of the bottom of the upper chamber was imaged using a BX53 upright fluorescence microscope (Olympus Corporation, Japan) after drying naturally. Cell counts in the upper chambers were obtained by thresholding for nuclear staining, followed by automated counting. Total cell counts were recorded as the sum of cell counts from the bottom of the upper and lower chambers. The cell migration index was calculated as the total area of migrating cells in the upper and lower chambers of the corresponding group divided by that of the WT CM group.

Acridine orange staining is an established technique for discrimination of bacterial cells and eukaryotic tissue in histological sections^{17 ,38} . Eukaryotic cells fluoresce green and prokaryotic cells fluoresce red-orange^{19 (link),38} . The staining response of bacteria, however, varies with physiology: active bacteria fluoresce red, and those in the stationary growth phase fluoresce green^{19 (link),39} . Sections of stained decayed tissue were stained using Acridine Orange using standard protocols^{17 ,38} and analyzed using an Olympus BX53 upright fluorescence microscope (×100 objective, N.A. 1.4) with filter cube sets for green (Exciter filter 470–495, DM505, Barrier filter 510–550 nm) and blue images (Exciter filter 540–550, DM570, Barrier filter 576–625 nm).