Alexa fluor 555

For immunofluorescence and/or flow cytometry the following antibodies were used: GK1.5 (anti-CD4), MP33 (anti-CD45), MECA-367 (anti-mucosal addressin cell adhesion molecule-1 [MAdCAM-1]), and ERMP-12 (anti-CD31), which were purified from hybridoma cell culture supernatants by affinity chromatography with protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) and labeled with Alexa Fluor-488, -555, or -647 (all from Invitrogen Life Technologies, Breda, the Netherlands). 145–211 (anti-CD3e, eBioscience, San Diego, CA, USA) Alexa Fluor-555 or Pe-labeled, and TUJ1 (anti-neuronal class III β-tubulin, BioLegend, Dedham, MA, USA) Alexa Fluor-488 labeled. Phage-display-derived single-chain antibodies vesicular stomatitis virus-tagged GD3A12 [11 (link)] and HS4E4 [12 (link)] were used and detected by a Cy3-conjugated secondary antibody anti-vesicular stomatitis virus, P5D4 (Sigma-Aldrich, St. Louis, MO, USA). To assure specificity of the antibodies, conjugate-alone controls as well as control serum (rat or rabbit) as replacement of the primary incubation were used. For western blots an anti-mouse decorin rabbit polyclonal was used at 1:1,000 dilution (immunogen: rat decorin; kindly provided by Åke Oldberg).

GFP-A. fumigatus germlings were propogated on coverslips, NK cells added at an MOI of 0.05 and co-incubated overnight, then washed twice with PBS and fixed and permeabilised in permeabilising solution 2 (BD Biosciences). Cells were washed with PBS, blocked in PBS containing 10% goat serum (2 h, room temperature) and incubated overnight at 4°C with a primary antibody (anti-LAMP-1, clone H4A3, Biolegend; anti-perforin, clone dG9, Biolegend; anti-granulysin, Santa Cruz) in blocking buffer. After washing with PBS, cells were incubated with anti-mouse Alexa Fluor® 555 (Biolegend) and Alexa Fluor® 633 phalloidin (Life Technologies) diluted 1:500 for 1 h at room temperature in the dark. Cells were washed with PBS and mounted with Vectashield mounting medium containing DAPI (Vector laboratories). Images were captured on a Zeiss LSM-510 confocal microscope.

HAE cells were differentiated for 7 weeks and then fixated in paraformaldehyde (4%) for 15 min at 37 °C. Cells were permeabilized with Triton X-100 (0.1%) for 5 min, blocked with BSA (1%), normal serum block (2%, Goat-serum, Biolegend) and Triton X-100 (0.05%) in PBS for 30 min. Subsequently, the cells were stained with mouse anti-β-tubulin-IV (30 μg/ml, Clone ONS.1A6, Sigma, staining ciliated cells) and rabbit anti-Muc5AC (3.37 μg/ml, Clone EPR16904, Abcam, staining mucus producing cells), or mouse anti-ZO-1 (10 μg/ml, Clone ZO1-1A12, staining tight junctions) for 1 h at 37 °C. For the secondary staining donkey anti-rabbit Alexa Fluor 555 (10 μg/ml, for Muc5AC staining, Biolegend), goat anti-mouse DyLight 488 (10 μg/ml, for β-tubulin-IV straining, Biolegend) or DyLight 649 (for ZO-1 staining, Biolegend) was added for 1 h at 37 °C. Nuclei were stained with DAPI (300 nM) for 10 min at 37 °C. The polyester membrane containing the cells was excised from the transwell insert, placed on a microscopy slide, and embedded in ProLong™ Diamond Antifade Mountant (Invitrogen). Images were acquired using the Leica Dmi8 microscope with a 100 × objective and Leica DFC7000 GT camera using LAS X 3.4.2 software.