Ultrastructural Localization of Antigens

Animals were anesthetized as above and perfusion fixed in 0.2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol-L^–1 PBS (pH 7.4). MA segments (∼2 mm in length) were washed (3 × 5 min) and processed in a Leica EMPACT 2 high-pressure freezer using 0.7% low melting agarose as a cryoprotectant. Samples were then freeze-substituted in a Leica AFS2 into 0.2% uranyl acetate in 95% acetone (from −85 to −50°C) and infiltrated with Lowicryl (at −50°C), before UV polymerization (2 days each at −50 and 20°C; Zechariah et al., 2020 (link)). Conventional transmission electron microscopy (TEM) was conducted using standard procedures (Sandow et al., 2002 (link), 2004 (link)).
Individual serial transverse sections (∼100 nm) were mounted on Formvar-coated slot grids and processed for antigen localization as for confocal immunohistochemistry (per above and Table 2). The secondary used was 5 or 10 nmol-L^–1 colloidal gold-conjugated antibody (1:40; 2 h) in 0.01% Tween-20. Sections were imaged at x10-40,000 on a JEOL transmission electron microscope at 16 MP (Emsis, Morada G3). Background gold label density was determined from randomly selected (4x) 1 × 1 μm regions per sample of lumen and IEL, compared to the same sized regions of interest in EC profiles.