Tecnai g2 spirit biotwin transmission em

Male mice at seven to eight months of age were deeply anesthetized using euthasol (pentobarbital; 150 mg/kg, i.p.). Following lack of response to toe pinch, mice were perfused transcardially with warm PBS (150 mm NaCl, 25 mm Sorensen’s PB; pH 7.4), then with warm fixative solution (4% PFA, 2.5% glutaraldehyde in 100 mm Sorensen’s PB pH 7.4) for 7–9 min. Brains were postfixed with 4% PFA in PB for 2–4 h. After overnight washing with PB, 100 μm thick coronal sections were cut on a Leica VT1200 vibratome. Sections were briefly washed with water, treated with 1% aqueous OsO₄ containing 1.5% potassium ferrocyanide for 40 min at 4°C, washed with water, followed by 1% aqueous OsO₄ for 1 h at 4°C. Slices were then en bloc stained with 1% uranyl acetate for 25 min at 4°C, dehydrated in an increasing series of ethanol, acetone, and propylene oxide, and flat embedded in Durcupan resin (Sigma-Aldrich); 70 nm thick sections were prepared using an EM UC7 Leica ultramicrotome, followed by counterstaining with 3% uranyl acetate and 0.5% lead citrate. Sections were examined in a Tecnai G2 Spirit BioTwin transmission EM (TEM; Thermo Fisher Scientific) at 100-kV accelerating voltage. A Veleta CCD camera (Olympus) operated by TIA software (Thermo Fisher Scientific) was used for image acquisition at a magnification of 20,500× and 60,000×, respectively.

Tissue samples for EM were harvested immediately after terminal anesthesia and immersion-fixed in 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 1 hour at room temperature. Tissue was post-fixed in 2% aqueous osmium tetroxide for 1 hour at 4°C. Tissue was rinsed in water, stained with 2% aqueous uranyl acetate overnight at 4°C, dehydrated in an ascending ethanol series, infiltrated with epon resin mixed with propylene oxide, and embedded in 100% epon resin. To determine the location of the carotid body, the polymerized sample was imaged using microCT (Zeiss Xradia 510 Versa) with a Zeiss filter LE2 and a ×4 objective lens. To confirm the presence of the carotid body by light microscopy, semithin sections were produced and stained using toluidine blue, as described previously (44 (link)). Ultrathin sections were produced and stained, as described previously (45 (link)). EM imaging was performed using a Tecnai G2 Spirit BioTWIN transmission EM (Thermo Fisher Scientific) equipped with a OneView CMOS camera (Gatan).