Lsm 700 axio examiner 2.1 confocal imaging system

Pig airway tissue was fixed in 4% formalin, embedded in paraffin, and cut in 4-μm-thick sections that were dewaxed using xylene substitute (Sigma) and hydrated. For immunohistochemistry, antigen retrieval was performed by microwave heating in 0.01 m citric buffer (pH 6). Sections were blocked for 60 min with Tris-buffered saline (TBS) with 3% donkey serum and 0.1% Triton X-l00. The primary antibody MUC5B (a kind gift from M. Kesimer, University of South Carolina, Chapel Hill, SC) in blocking solution was incubated overnight at 4 °C and washed in TBS three times for 5 min before adding donkey anti-rabbit Alexa 555 (Thermo Fisher Scientific, Waltham, MA). Nuclei were counterstained with Hoechst 34580 (Thermo Fisher Scientific). Slides were washed in TBS three times for 5 min and mounted with Prolong Gold mounting medium (Thermo Fisher Scientific). Images were acquired with an upright LSM 700 Axio Examiner 2.1 confocal imaging system (Carl Zeiss, Oberkochen, Germany).

Newborn WT piglet tracheas were dissected as for video microscopy into 1 cm pieces, opened and pinned with insect pins to Petri dishes coated with Sylgard. Mucus was stained with fluorescein labeled Lotus tetragonolobus (LTL) lectin and/or DyLight 649 labeled Ulex europaeus Agglutinin I (UEA1) mixed in oxygenated Krebs-glucose buffer at 5 µg/ml and added to the tissue. Nuclei were stained with Syto 59 at 20 µM (Cat# S11341, ThermoFisher Scientific, Waltham, MA). The tissue was washed after approximately 30 min of incubation at ambient temperature in the dark and 2 ml fresh oxygenated Krebs-glucose buffer was added. Mucus threads were visualized with a Plan-Apochromat × 20/1.0DIC water immersion objective (Cat# 420762-9900-000, Carl Zeiss, Oberkochen, Germany), an upright LSM 700 Axio Examiner 2.1 confocal imaging system (Zeiss LSM 700 microscope, RRID:SCR_017377) or a Zeiss LSM900 Axio Examiner Z1 microscope with the Airyscan 2 imaging system and Zen blue software (RRID:SCR_013672, Carl Zeiss, Oberkochen, Germany). After acquisition, Airyscan images were processed using standard Airyscan processing algorithms.