Stx 6 polyclonal antibody

First, the total spore protein of mature spores was extracted (59 (link)). Concisely, mature spores (1 × 10⁹ spores/mL) were disrupted for 5 minutes for three times with 0.4-g acid-washed glass beads (0.2 g 212–300 μm and 0.2 g 425–600 μm; Sigma-Aldrich, St. Louis, USA) in 500-µL buffer PBS, pH 7.4, containing PMSF by Bioprep-24 Homogenizer (Beijing, China) and then centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant was collected as the spore total protein samples. For immunoblotting analysis, 10-µg protein samples were subjected to SDS-PAGE, and the gel was transferred to the PVDF membrane (Roche, Shanghai, China). After blocking in 5% skim milk diluted in TBST (150 mM NaCl, 20 mM Tris-HCl, 0.05% Tween-20), the membrane was incubated with STX 6 polyclonal antibody (diluted 1:1,000 in blocking solution, ImmunoWay, Jiangsu, China) for 1.5 hours at 37°C. The negative rabbit antiserum was used as a negative control. Then, the membrane was washed and incubated with goat anti-rabbit IgG (1:8,000 dilution; Sigma-Aldrich, St. Louis, USA) for 1 hour at 37°C. Washing was performed three times with TBST, and the blot was developed with ECL Western blot detection kit (Thermo Fisher Scientific, Shanghai, China).

Ultrathin sections (70 nm) of mature spores were prepared as previously described (60 (link)) and placed on nickel grids (Quantifoil, Beijing, China). After blocking with 10% (vol/vol) non-specific goat serum together with 5% (wt/vol) BSA in PBST at room temperature for 1 hour, the grids were incubated with STX 6 polyclonal antibody (1:50 dilution; ImmunoWay, Jiangsu, China) or the negative rabbit antiserum at room temperature for 1 hour. The grids were then incubated with gold-conjugated anti-rabbit IgG (1:30 dilution; Sigma-Aldrich, St. Louis, USA). Then, the samples were stained in 3% (wt/vol) uranyl acetate (Zhongjingkeyi Technology Co., Ltd., Beijing, China), followed by 1% (wt/vol) lead citrate (Zhongjingkeyi Technology Co., Ltd., Beijing, China). Samples were observed using the Hitachi HT7800 electron microscope (Hitachi, Tokyo, Japan) at 80 kV.