Eponate 12 kit with dmp 30

Transmission electron microscopy was performed under a standard protocol as previously described (71 (link), 72 (link)). In brief, tracheas were dissected, and lung tissues were obtained from WT mice and Pycr1-deficient mice that were exposed to HDM or PBS for 5 weeks. Fresh tissues (approximately 1 mm × 1 mm × 1 mm in size) from the same location of the lung were quickly soaked in 2.5% glutaraldehyde in 0.1 M phosphoric buffer (pH: 7.4) for 24 hours and postfixed in 1% osmium tetroxide for 2 hours at RT in the dark. After removal and rinsing, the samples were dehydrated with gradient alcohol, infiltrated, and embedded using an Eponate 12 Kit with DMP-30 (TED PELLA Inc, 18010). The blocks were cut into 100 nm–thick sections on an ultramicrotome, stained with lead citrate (2.6%) to prevent CO₂ staining, and counterstained with uranium acetate. The copper grids were observed under a Hitachi transmission electron microscope (JEM-1400Flash).

Temporal bones were dissected, and a small hole in the temporal bone was generated adjacent to the oval window. Fixative (2% paraformaldehyde/3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4) was injected into the hole, and temporal bones were immersion-fixed overnight at 4°C. Utricles were removed, and the otoconial membrane and otoconia were dissected away. Utricles were post-fixed in 1% osmium tetroxide and embedded in Eponate 12 Kit with DMP-30 (Ted Pella Inc., Redding, CA). Semi-thin sections (2 µm) were mounted onto gel-coated slides and stained with toluidine blue dye [Sigma-Aldrich (St. Louis, MO)]. Sections were imaged using a Zeiss Axioplan microscope (Zeiss, Jena, Germany).