Anti e coli antibody

E. coli (MCLAB) and S. flexneri (Sigma-Aldrich) were inoculated into a Luria-Bertani broth and nutrient broth, respectively, and cultured overnight in a 37 °C incubator. Bacteria cells were pelleted by centrifugation and then resuspended in phosphate-buffered saline (PBS). The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 10% bovine serum albumin in PBS. Anti-E. coli antibody (Abcam, goat polyclonal antibody) and anti-S. flexneri antibody (Bio-Rad, mouse monoclonal antibody) were labelled by Mix-n-Stain biotin (Sigma-Aldrich). The labelled antibodies were purified with an ultrafiltration membrane column and centrifuged at 14,000 × g. Then, the bacteria cells were incubated with the biotin-labelled antibodies for 1 h at room temperature. The bacteria concentration was 4 × 10⁷ cells per ml. Streptavidin-coated silica microparticles (Bangs laboratories, Inc.) with a diameter of 0.99 μm were suspended in PBS and the concentration was adjusted to 4 × 10⁷ particles per ml.

Paraffin-embedded sections were cut 5 μm thick, mounted on precoated hydrophilic glass slides (Matsunami Glass), dried at 37°C to ensure adherence to the slides, and stored at 4°C until use. Sections were baked at 50°C overnight before staining. Paraffin sections were deparaffinized in 3 changes of xylene for 10 min each, then rehydrated through a series of graded alcohols with a final rinse in distilled water. Sections were then subjected to antigen retrieval by heating at 121°C in citrate buffer for 20 min. The slides were then rinsed in distilled water, TBS, and blocked with 3% BSA/TBS-T for 1 h at RT. Then, tissues were stained with anti—E. coli antibody (Abcam, Cambridge, MA, USA, Cat. # ab137967, 1:1000 df), anti-Ly-6G/Ly-6C antibody (RB6-8C5) (Novus Biologicals, Littleton, CO, USA, Cat. # NBP2-00441, 1:100 df) and anti-TLR-4 antibody (Novus Biologicals, Littleton, CO, USA, Cat. # NBP2-24821, 1:200 df) diluted in 3% BSA/TBS-T overnight at 4°C. The next day, the tissues were washed then incubated with secondary antibody (Alexa Fluor® 594, Abcam, Cat# ab150080, Lot: GR3323881-1 with 1:1000 df for E. coli, TLR-4, and DyLight 650, Novus Biologicals, Cat # NBP2-60688C, Lot: 39933-102120-c with 1:1000 df for Ly-6G/Ly-6C) for 2 h at RT followed by DAPI staining. Images were obtained and analyzed as described above using the BZ-X800 Analyzer (Keyence Corp).