Immunostaining of Pathogenic Bacterial Infections

For immunostaining, monolayers were fixed in 4% paraformaldehyde at room temperature for 15 min, followed by storage in 70% ethanol at 4°C until paraffin embedding (33 (link)). Embedding and sectioning were performed by the Specialized Histopathology Core of Massachusetts General Hospital. Prior to staining, sections were deparaffinized using xylene with gradual rehydration in decreasing concentrations of ethanol. Sections were blocked using 0.4% goat and donkey serum in 0.04% Triton X-100 in PBS. Sections were stained using the antibodies against actin (3700S; Cell Signaling Technologies), Mucin 2 (sc-13312; Santa Cruz Technologies) (33 (link)), Salmonella (8209-4006; Bio-Rad), and E. coli (ab137967; kind gift of Deepak V. K. Kumar; Abcam). Fluorescently conjugated secondary monoclonal antibodies (Alexa Fluor 488- and 555-conjugated antibody series against mouse, rabbit, or goat from Life Technologies) were used for detection. Nuclei were counterstained with 6-diamidino-2-phenylindole (DAPI). Samples were imaged using a Nikon A1SiR confocal microscope.