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).
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