Antifade reagent

The harvested samples were fixed with PFA for 30 min and then soaked overnight in 30% sucrose solution at 4°C on a nutating tube rocker. Next, they were immersed in optimal cutting temperature compound (23-730-571, Fisher Scientific) and placed in a cryostat set at −20°C. Cryosections of 40- or 60-μm thick were made and placed on poly-l-lysine (0.1%, w/v)–coated slides.
The cryosectioned samples were gently washed with DPBS, permeabilized with 0.1% Triton X-100 (T8787, Sigma-Aldrich), and blocked with 2% bovine serum albumin (A2153, Sigma-Aldrich). Primary rabbit VE-cadherin antibody (2158, Cell Signaling Technology) was diluted 1:200 in cell staining buffer (420201, BioLegend) and incubated with the samples at 4°C overnight. The primary antibody was then labeled with donkey anti-rabbit immunoglobulin G CF543 secondary antibody (20308-1, Biotium), which was diluted in cell staining buffer at 1:200 and incubated at 37°C for 2 hours. Cytoskeleton and nuclei were labeled with Phalloidin eFluor 660 (50-6559-05, eBioscience) and DAPI (4083S, Cell Signaling Technology) per the manufacturer’s instruction before the slides were mounted with antifade reagent (9071S, Cell Signaling Technology).

After exposure of formalin-fixed HCT-8 cell monolayers to viable or dead S. Enteritidis (1 × 10⁸ cells/ml) for 30-min, the wells of the chambered slides (Fisher Scientific) were washed with PBS to remove unattached bacterial cells (as above). After immunoprobing with mAb-2F11, the monolayers were washed and probed with Alexa Fluor 488 conjugated anti-mouse antibody for 1.5 h at room temperature in the dark, followed by three PBS wash. Note, antibody concentrations used were the same as above. The monolayers were counterstained with DAPI (500 ng/mL; Cell-Signaling) for nuclear staining and the slides were mounted using an antifade reagent (Cell-Signaling). Images were acquired using the Nikon A1R confocal microscope with a Plano AP VC oil immersion objective (Drolia et al., 2018 (link)) and were processed with the Nikon Elements software at the Purdue Bindley Bioscience Imaging Facility.
For Giemsa staining, the formalin-fixed HCT-8 cell monolayers were exposed to viable or dead S. Enteritidis cells as above, air-dried, and immersed in Giemsa staining solution for 20 min. Giemsa staining solution was prepared using a 20-fold dilution of the KaryoMAX Giemsa staining solution (Thermo-Fisher) in deionized water. The slides were examined under a Leica DAS Microscope at the magnification of 1,000×.