Msp 2s

For scanning electron microscopy analysis, the strains were cultured in NA medium containing 150 mg/L Pb²⁺ for 12 and 24 h, respectively. The control comprised NA medium without Pb²⁺. A total of 10 mL of the culture broth were centrifuged at 2292 × g for 10 min, washed with sterile water, and centrifuged again at 5000 rpm for 10 min to collect the cells. The cells were suspended in 40-fold volume of 2.5% glutaraldehyde fixative, stored in a refrigerator for more than 24 h at 4 °C, rinsed with phosphate buffer for 2–3 times, and subjected to gradient dehydration with 50%, 70%, 90%, and 100% ethanol, respectively. After dehydration, the cells were treated with ethanol-tert-butanol solution (V1:V2 = 1:1) for 20 min, and with 100% tert-butanol twice for 20 min each. Subsequently, the samples were freeze-dried, sputter-coated using ion sputtering equipment (IXRF SYSTEMS, MSP-2S, Japan), and observed and photographed under a scanning electron microscope (Hitachi, S3400N, Japan).

SS1 was inoculated into Brucella broth containing 5% (vol/vol) FBS and cultured with LipoLLA (7.5 μg/mL) for 24 h. Then, the bacteria were harvested by centrifugation at 3000 rpm for 10 min, and the contents of GLU and AST in the supernatant were detected by an automatic biochemical analyzer (HITACHI, Japan) to study the effect of LipoLLA on the outer membrane barrier of H. pylori. The remaining pellets treated with LipoLLA (7.5 μg/mL) were prepared for observing the ultrastructure of the strain by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Briefly, pellets were fixed with 2% glutaraldehyde for 2 h at room temperature. The SEM samples were post fixed with 1% osmium acid in phosphate buffer (0.1 M, pH 7.4), dehydrated, dried by critical point drier (Quorum, K850, UK), and then sprayed with gold over 30 s by carbon coater (IXRF, MSP-2S, USA) before SEM (HITACHI, SU8100, Japan) imaging. To prepare the TEM samples, the fixed bacteria were resuspended in 1% agarose after centrifugation, then post fixed with 1% osmium acid in phosphate buffer (0.1 M, pH 7.4) for 2 h; After dehydration, infiltration and embedding, ultramicrotome (Leica, Leica UC7, Germany) was used for slicing to 60-80 nm sections, and then dried overnight after double-staining with uranium lead. Finally, sections were examined by TEM (HITACHI, HT7700, Japan).

The fungal hyphae in the infected leaf tissues were harvested and cleared in the Carnot fixator overnight at room temperature. The samples were then rinsed and stained with WGA-Alexa Fluor 488 (Invitrogen, CA, USA), and the plant membrane was visualized using propidium iodide (PI), as described in an earlier study (Gao et al., 2013 (link)). The samples were then observed under a confocal microscope (Zeiss, LSM 780, Germany).
Scanning electron microscopy (SEM) was carried out to observe the infection dynamics of samples collected at different time points after inoculation. The samples were fixed in glutaraldehyde for 2 h and continuously dehydrated with alcohol and tert-butanol, followed by freeze-drying (Vacuum Device, VFD-30, Japan). These samples were coated with gold powder by spraying (Hitachi, MSP-2S, Japan) and observed using an SEM (Hitachi, TM-3030, Japan).
Furthermore, the leaf samples were analyzed by transmission electron microscopy (TEM) to determine the subcellular structural differences. The samples were fixed overnight in glutaraldehyde and dehydrated in acetone. These light microscopy and electron microscopy samples were processed as described by Redkar et al. (2015 (link)). Ultrathin sections were made using a Leica ultramicrotome (Leica, EM UC7, Germany) and observed under a Hitachi TEM (Hitachi, HT7700, Japan).