Immunofluorescence Analysis of IL-25 and IL-17RB

We have previously confirmed the differentiation status (stratified, active cilia, mucin-positive goblet cells) of the ALI-BEC cultures from which sections were subsequently obtained for immunofluorescence-based analyses of IL-25 and IL-17RB expression for this study^{25 (link)}. Paraffin-embedded endoscopic lung biopsies and ALI-BEC sections were deparaffinised in xylene, then rehydrated in ethanol before being subjected to antigen retrieval in sodium citrate buffer. Slides were washed in TBS-T then were blocked for 1 h in 5% donkey serum/5% casein solution in TBS-T in a humidified chamber. The blocked sections were incubated overnight at 4 °C with anti-IL-17RB (MAB1207, R&D Systems) and anti-IL-25 (BAF1258, R&D Systems, USA) in 2% donkey serum/2% casein in TBS-T. Primary antibodies were washed off and HRP-488 (405235, Biolegend, USA) and Alexa-fluor-594 (SAB4600407, Sigma Aldrich, USA) were applied in the dark for 2 h at room temperature. Secondary antibodies were washed off and mountant containing DAPI was applied prior to imaging with the Axio imager M2 fluorescent microscope and analysing with Zen 2 software (Carl Zeiss AG, Germany).