Nasal brush biopsies were obtained from the middle turbinate with a cytology brush and suspended in RPMI cell culture medium (GIBCO) (Wallmeier et al., 2014 (link)). Cells were spread on glass slides, air dried and stored at −80°C until use. Defrosted samples were fixed in 4% PFA in PBS for 15 minutes. After washing with PBS, the cells were permeabilized with 0.2%Triton-X 100 in PBS for 15 minutes. Following washes with PBS, samples were blocked in blocking solution (1–5% skim milk in PBS) at 4°C overnight. Samples were incubated for at least 3 hours with primary antibodies diluted in blocking solution. Slides were rinsed with PBST, washed with blocking solution and incubated with secondary antibodies in blocking solution for 30 minutes. After washes with PBS, the nuclei were stained with Hoechst 33342 for 10 minutes. High-resolution fluorescence images were acquired either with a Zeiss LSM880 using ZEN2 software or with a Zeiss AxioObserver Z1 Apotome with AxioVision 4.8 software (Carl Zeiss, Jena, Germany) and processed with ZEN2 Software and Adobe Creative Suite 4 (Adobe Systems Incorporated, San Jose, CA).
All other images were acquired on the Leica TCS SPE laser scanning confocal microscope and processed using FIJI and Adobe Photoshop CS5.1 unless otherwise specified.
All other images were acquired on the Leica TCS SPE laser scanning confocal microscope and processed using FIJI and Adobe Photoshop CS5.1 unless otherwise specified.