S 3400n vp sem microscope

Adult mouse airway tissues were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4 (all from Electron Microscopy Sciences) at 4°C overnight, osmicated, dehydrated and dried with a Tousimis Autosamdri-815 critical point dryer. Samples were then mounted luminal side up, sputter coated with 100 Å layer of Au/Pd and analyzed with a Hitachi S-3400N VP-SEM microscope (Hitachi) operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.

For wholemount immunofluorescence, HNECs were fixed in −20°C methanol or 4% paraformaldehyde for 10 min as previously described (31 (link)). Transwell filters were cut out of the plastic supports and placed in a humid chamber for staining. Samples were blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1–2 h, then with Alexa dye conjugated secondary antibodies (Thermo Fisher) for 30 min at room temperature. Filters were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma). Samples were imaged with a Leica SP8 or Zeiss LSM 900 confocal microscope. For antibodies and fixation conditions, see Supplemental Table S1. For SEM, Transwell filters were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M NaCacodylate buffer, pH 7.4 at 4°C overnight. Samples were osmicated, dehydrated, dried with a Tousimis Autosamdri-815 critical point dryer. Samples were mounted luminal side up, sputter coated with 100 Å layer of Au/Pd. Images were acquired with a Hitachi S-3400N VP-SEM microscope operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.