Image it fixative solution

Sub-cellular localization of BDP FL-labelled cheopin was determined using confocal microscopy. Briefly, cells (1×10⁸ CFU/mL) were stained with 1 μM aldehyde-fixable membrane-binding dye FM4–64 FX (Thermo) in PBS for 10 min at room temperature and the unbound dye was removed by washing 2 times with 1 mL PBS by centrifugation (5 min, 7000 × g). Cells were then incubated with 2 μM of BDP FL-cheopin in PBS for 10 min at room temperature and subsequently washed with PBS as in the cell binding assays to remove unbound peptide. Peptide-treated cells were fixed overnight at 4°C in Image-iT fixative solution (Thermo). Fixed cells were washed with 1 mL of PBS (5 min, 10000 × g), and cells were resuspended in 20 μl of ProLong glass antifade mountant (Thermo) and spotted on a cover glass. Control samples treated with 2μM free BDP FL were used to rule-out any non-specific interaction with Y. pestis cells. Images were recorded on a Leica SP8 Lightning confocal microscope with a 63X oil immersion objective (NA 1.4). BDPFL and FM4–64 were excited using Argon 488 nm and HeNe 561 nm lasers, with emission signals collected at 510–550 nm and 640–740 nm, respectively. Images were processed for brightness, contrast, and quantification of signal using LAX software (Leica Microsystems).

Phase contrast micrographs of live cell morphology were acquired with an inverted microscope (Olympus) right before device assembly and daily after assembly for 5 days. Cells were then analyzed by immunofluorescence staining for VE-cadherin and actin filaments (F-actin). Staining was carried out at room temperature. Cells were fixed with Image-iT™ Fixative Solution (4% paraformaldehyde, Thermo Fisher) for 10 min, washed with DPBS (Thermo Fisher), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in DPBS for 10 min, blocked with 5% bovine serum albumin (BSA) blocking buffer (Alfa Aesar, Haverhill, MA) for 1 hour, and then incubated with Alexa Fluor 488 conjugated VE-cadherin monoclonal antibody (4 µg/mL, Santa Cruz Biotechnology, Dallas, TX) and Cruzfluor 555 conjugated phalloidin (1 µg/mL, Santa Cruz Biotechnology) in 1% BSA for 2 hours. Samples were then washed 3 times in DPBS and mounted on slides with Fluoroshield™ with DAPI (Sigma-Aldrich) for nuclear counterstain. Images were captured with a Zeiss LSM 710 confocal microscope and analyzed in ImageJ. Visual orientation analysis on cell actin filaments was performed using an ImageJ directional analysis plugin – OrientationJ^{14 (link)}. Cells were counted based on nuclear staining.