Zen 2.3 pro

U2OS and HeLa cells were grown on coverslips up to 50% confluence. The cells were washed with chilled PBS and fixed with 4% paraformaldehyde in PBS. The cells were permeabilized with 0.15 v/v% Triton-X 100 for 3 min. Nonspecific sites were blocked with 2% bovine serum albumin (Sigma-Aldrich) in PBS for 45 min. Fixed cells were stained with the following antibodies (for 3 h at 37 °C): β-Tubulin (1:100, Sigma-Aldrich), DBC1 (1:100, Cell signaling), and HA (1:100, Invitrogen). Primary antibodies were visualized by secondary antibodies conjugated to Alexa fluor 488 and Alexa fluor 594 (1:5000, Jackson ImmunoResearch) at 37 °C for 1 h. The cells were stained with DAPI (BioRad) for 10 min. Pictures were taken with an inverted fluorescence microscope (Axio Observer, Colibri7, Carl Zeiss MicroImaging, Inc) using filters for DAPI, GFP and mRF12, X20, X63, and DIC Apotome.2 oil objectives. The pictures were generated with ZEN 2.3 pro (Zeiss).

EVPL tissue pieces infected with P. aeruginosa, and uninfected tissue negative controls, were fixed, paraffin-embedded, de-paraffinized and re-hydrated as described for H & E staining. Slides were stained in 1% Alcian blue solution (pH 2.5) (Sigma-Aldrich) then rinsed using distilled water. The samples were counterstained in nuclear fast red solution (Alfa Aesar) and rinsed briefly in distilled water. Tissue sections were dehydrated in increasing ethanol concentrations and finally placed in xylene, as performed for H & E staining. Samples were mounted with DPX mounting fluid and images were taken of stained tissue sections using a Zeiss Axio Scope. A1 light microscope with the Zeiss Axiocam Erc 5s and Zeiss Zen 2.3 pro software.

P. aeruginosa infected EVPL tissue pieces, plus uninfected controls, were fixed and paraffin-embedded as described above for H & E staining. The tissue sections were de-paraffinized in xylene. Samples were re-hydrated in 100% (v/v) ethanol for then moved to fresh 100% (v/v) ethanol. Slides were placed in 95% (v/v) isopropanol then 70% (v/v) isopropanol. Residual alcohol was removed by washing slides in distilled water. Crystal violet (Pro-Lab Diagnostics) was applied to the samples and rinsed with water, then iodine (Pro-Lab Diagnostics) was applied. The iodine was then rinsed from the samples using tap water and acetone added for decolourisation. Tap water was used to rinse off the acetone and 1% (v/v) neutral red (Pro-Lab Diagnostics) applied to counterstain. The slides were again rinsed with tap water and blotted dry with filter paper. The samples were dehydrated in 100% (v/v) isopropanol then placed in two fresh changes of 100% (v/v) xylene. Samples were mounted using DPX mounting fluid and images taken using a Zeiss Axio Scope. A1 light microscope with the Zeiss Axiocam Erc 5s and Zeiss Zen 2.3 pro software.