Anti α sma fitc 1a4

Lung or intestinal sections were incubated with IHC blocking buffer (PBS, 0,3 % v/v Triton X-100 (Thermo Fisher Scientific Inc.), 10 % v/v normal mouse serum (Invitrogen Inc.)) for 20 min at RT. Sections were incubated in IHC blocking buffer with purified anti-RORγt (AFKJS-9; Invitrogen Inc.) and where indicated with anti-α-SMA-FITC (1A4; Invitrogen Inc.), anti-TCRβ-AF647/AF488 (H57-597; BioLegend Inc.) or anti-CD11c-AF647 (N418; BioLegend Inc.) diluted in PBS (overnight (intestinal sections) or 60 h (lung sections), 4°C). After three washing steps, sections were incubated with secondary polyclonal anti-rat IgG-DyLight-549/AF647 (Jackson ImmunoResearch Inc.) in PBS supplemented with 5 % normal mouse serum (Invitrogen Inc.) for 60 min at RT. For double-labeling with CD3, an additional blocking step with 10 % normal rat serum (Linaris Biologische Produkte) was performed for 1h at RT, followed by incubation with anti-CD3-AF647 (17A2; BioLegend Inc.) for 2h, at RT. Slices were rinsed three times and cover slipped with Mowiol mounting medium (12 g Mowiol 4-88 (Sigma-Aldrich), 30 mL A. bidest., 60 mL Tris buffer (0.2 M; Invitrogen Inc.), 30 g Glycerin (pH 8.5; Merck)) with or without 1 µg/mL Hoechst dye (Thermo Fisher Scientific Inc.).

Stained slices were evaluated with confocal microscopy. Z-stacks (40 μm with a resolution of 1 μm) were recorded to allow a three-dimensional presentation of lung structures including possible cell interactions. Airway and blood vessels were identified by the use of anti-α-SMA-FITC (1A4; Invitrogen) or using autofluorescence (excitation: 488nm; detection: 490-530 nm). Analysis was performed using a laser scanning confocal microscope Zeiss 710 meta (Carl Zeiss AG).