Mouse anti muc5ac

Intact ALI culture inserts were fixed for 15 min at room temperature in 3.2% paraformaldehyde. Histology sections were stripped of paraffin using HistoChoice Clearing Agent (Sigma), rehydrated using a decreasing gradient of alcohol washes (100%, 90% 70%, 50%, 30%, 0%), and antigen retrieval conducted using Citric Acid-based Antigen Unmasking Solution pH 6.0 (Vector Laboratories). Prior to labeling, all samples were blocked and permeabilized for 30 min using 3% BSA/0.1% Triton X-100 in tris-buffered saline (TBS). Primary labeling was conducted using 3% BSA/0.1% Triton X-100 in TBS for 1 h with mouse anti-MUC5AC (1:500; ThermoScientific). Sections were washed twice using TBS/0.1% Triton X-100 (TBST) and secondary labeling was conducted using DAPI (1:1000) and AlexFluor594 goat anti-mouse IgG (1:500; ThermoScientific) for 30 min. Slides were washed twice in TBST and mounted with Vectashield HardSet Antifade Mounting Media (Vector Laboratories). Images were acquired using a Revolve microscope (Echo Labs) at 10x magnification.

Near-native sections were removed from any remaining agarose using forceps and subsequently transferred to a 12-well plate into wells containing blocking and permeabilization solution (5% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) and incubated O/N (∼18 h) at 4°C with shaking. The following day, the blocking and permeabilization solution was replaced with primary antibody diluted in blocking solution (1% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) and the section incubated for 72-96 h at 4°C. The following primary antibodies were used: rabbit anti-Ki67 (1:250; A. Menarini, MP-325-CRM1), Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), rabbit anti-H+/K+ ATPase β Antibody (D-18) (1:300; Santa Cruz Biotechnology, sc-84304), mouse anti-MUC5AC (1:300; Thermo Fisher Scientific, MA512178), Alexa 488-conjugeted Lectin GS-II (1:500; Thermo Fisher Scientific, L21415). Sections were subsequently washed and incubated with an appropriate secondary antibody and DAPI in blocking solution (1% DMSO, 0.5% Triton X-100 and 2% NDS in PBS) for 48 h at room temperature with shaking. After washing for 3 × 45 min with PBS, sections were carefully transferred from wells to microscope slides using a brush before mounting in RapiClear 1.52 (CamBioScience). Slides were sealed and stored at 4°C in the dark before imaging by fluorescent microscopy.