Female C57BL/6 mice (6–8 weeks) were bred and maintained at the experimental animal center of Soochow University. All animal experiments were approved by the Ethics Committee of Soochow University and conducted in accordance with the Guidelines for the Care and Use of Research Animals established by Soochow University. The streptomycin-pretreated mouse model was established as described previously.40 (link) Mice that fasted for 4 h were administered intragastrically with streptomycin (100 μl of 200 mg/ml solution in sterile water; Sigma, USA). After 24 h, mice were randomly divided into four groups, including control, STM-WT, STM-ΔsopF, and STM-ΔsopF/psopF infection groups. 100 µL of either 5 × 107 colony-forming unit (CFU) S. Typhimurium strains or sterile PBS were used for oral gavage to mice that were fasted for 4 h before infection. Mice were sacrificed using CO2 asphyxiation at indicated time points.
For histopathology, tissue samples were harvested and fixed in 4% paraformaldehyde, processed according to standard procedures for dehydration, paraffin embedding, section cutting, and deparaffinization. The sections were stained with hematoxylin–eosin (Baso, Zhuhai, China) and observed under a light microscope (Olympus, Japan). Total inflammatory scores were assessed based on the following parameters according to previous literature:28 (link) neutrophil infiltration (0, none; 1, slight increase; 2, marked increase), fibrin deposition, submucosal neutrophil margination, submucosal edema, epithelial necrosis, epithelial ulceration (0, absent; 1, present). The percentage of pathological lesions was counted on a total scale of 0–20.
For bacterial burden measurement, spleen, and liver were harvested and then immersed in 100 µg/ml of amikacin for 1 h. Tissues were homogenized in PBS containing 0.3% Triton (Sigma, USA) for 30 min. CFU values were quantified by plating lysates with appropriate dilutions onto Salmonella-Shigella agar (Hangwei, China), followed by incubation overnight.
For immunofluorescence analysis, ceca samples were fixed with 4% paraformaldehyde at 4°C overnight, processed for paraffin embedding. Tissues were cut into 6-μm transverse sections, followed by deparaffinizing and rehydrating for antigen retrieval. The sections were blocked with 3% BSA for 30 min and then incubated with diluted primary antibodies at 4°C overnight. The antibody against EpCAM (#GB11274) was purchased from Servicebio (Wuhan, China; 1:3000 dilution). The antibody against S. Typhimurium O antigen (#S10820100) was purchased from the Lanzhou Institute of Biological Products Co., Ltd. (Lanzhou, China; 1:200 dilution). Next, the slides were incubated with Cy3 (#GB21303; 1:300 dilution) or Alexa Fluor 488 (#GB25303; 1:400 dilution) conjugated goat anti-rabbit immunofluorescent secondary antibodies purchased from Servicebio (Wuhan, China). The nuclei were stained with DAPI (#G1012) purchased from Servicebio (Wuhan, China). Slides were mounted in Anti-fade mounting medium (#G1401; Servicebio). Images were photographed using a Nikon microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan).
Sample processing for transmission electron microscopy (TEM) was carried out in the School of Biology and Basic Medical Science, Medical College of Soochow University. Ceca samples were fixed in ice-cold 2.5% glutaraldehyde for at least 4 h. Samples were washed twice using 0.1 M phosphate buffer for 15 min at room temperature. Subsequently, the samples were post-fixed in 1% OsO4 for 1 h, dehydrated through an acetone series and embedded in epoxy resin. Ultra-thin sections were stained and observed using a 120 kV Transmission electron microscope (HT7700, Hitachi, Japan).
For histopathology, tissue samples were harvested and fixed in 4% paraformaldehyde, processed according to standard procedures for dehydration, paraffin embedding, section cutting, and deparaffinization. The sections were stained with hematoxylin–eosin (Baso, Zhuhai, China) and observed under a light microscope (Olympus, Japan). Total inflammatory scores were assessed based on the following parameters according to previous literature:28 (link) neutrophil infiltration (0, none; 1, slight increase; 2, marked increase), fibrin deposition, submucosal neutrophil margination, submucosal edema, epithelial necrosis, epithelial ulceration (0, absent; 1, present). The percentage of pathological lesions was counted on a total scale of 0–20.
For bacterial burden measurement, spleen, and liver were harvested and then immersed in 100 µg/ml of amikacin for 1 h. Tissues were homogenized in PBS containing 0.3% Triton (Sigma, USA) for 30 min. CFU values were quantified by plating lysates with appropriate dilutions onto Salmonella-Shigella agar (Hangwei, China), followed by incubation overnight.
For immunofluorescence analysis, ceca samples were fixed with 4% paraformaldehyde at 4°C overnight, processed for paraffin embedding. Tissues were cut into 6-μm transverse sections, followed by deparaffinizing and rehydrating for antigen retrieval. The sections were blocked with 3% BSA for 30 min and then incubated with diluted primary antibodies at 4°C overnight. The antibody against EpCAM (#GB11274) was purchased from Servicebio (Wuhan, China; 1:3000 dilution). The antibody against S. Typhimurium O antigen (#S10820100) was purchased from the Lanzhou Institute of Biological Products Co., Ltd. (Lanzhou, China; 1:200 dilution). Next, the slides were incubated with Cy3 (#GB21303; 1:300 dilution) or Alexa Fluor 488 (#GB25303; 1:400 dilution) conjugated goat anti-rabbit immunofluorescent secondary antibodies purchased from Servicebio (Wuhan, China). The nuclei were stained with DAPI (#G1012) purchased from Servicebio (Wuhan, China). Slides were mounted in Anti-fade mounting medium (#G1401; Servicebio). Images were photographed using a Nikon microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan).
Sample processing for transmission electron microscopy (TEM) was carried out in the School of Biology and Basic Medical Science, Medical College of Soochow University. Ceca samples were fixed in ice-cold 2.5% glutaraldehyde for at least 4 h. Samples were washed twice using 0.1 M phosphate buffer for 15 min at room temperature. Subsequently, the samples were post-fixed in 1% OsO4 for 1 h, dehydrated through an acetone series and embedded in epoxy resin. Ultra-thin sections were stained and observed using a 120 kV Transmission electron microscope (HT7700, Hitachi, Japan).
Free full text: Click here
