Leica sp8 inverted confocal microscope

Tissues were processed, embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin (H&E) as previously described [72 (link)]. Sections were used for IF imaging of P. aeruginosa exotoxin A and ZO-1 using the following antibodies: goat anti-exotoxin A (1:500; #LS-C50940, LifeSpan Biosciences; Seattle, WA, USA); donkey anti-goat AlexaFluor^®® 568 (1:1000; #A-11057, ThermoFisher; Waltham, MA, USA); rabbit anti-ZO-1 (1:100; #40-2200, ThermoFisher; Waltham, MA, USA); goat anti-rabbit AlexaFluor^®® 488 (1:1000; #A11008, ThermoFisher; Waltham, MA, USA). All sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 175 µg/mL; #D1306, ThermoFisher; Waltham, MA, USA) to visualize nuclei. All images were captured using Leica Inverted Confocal Microscope SP8 (Leica Microsystems) and saved as .tif images.

The pcDNA-ER-ZapCY1 vector was generated by Dr. Amy Palmer^{52 (link)} and purchased from Addgene (Cambridge, MA). ER-ZAPCY1is a high affinity Zn sensor targeted to the ER that is sandwiched between two fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Zinc binding induces a conformational change leading to an increase in fluorescence resonance energy transfer (FRET) from CFP to YFP^{52 (link)}. To first confirm the localization of ER-ZAPCY1in our system, MECs were plated on glass coverslips in a 24-well plate and transfected with ER-ZAPCY plasmid (0.8 µg/well) using Lipofectamine 2000 as described above. After 24 h, MECs were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and then ER-ZAPCY1(anti-GFP antibody; 1:50, Sigma-Aldrich) and calnexin (1:50; Abcam) were detected. Antibodies were visualized with Alexa Fluor® 488 or Alexa Fluor® 568 (Life Technologies) and counterstained with DAPI nuclear stain (1 µg/mL). Slides were examined using the Leica Inverted Confocal Microscope SP8 (Leica Microsystems). In subsequent experiments, MECs were co-transfected with T²⁸⁸ or S²⁸⁸ and ER-ZAPCY1 as described above, and ER-ZAPCY1 and ZnT2-HA (using anti-HA antibody; 1:100, Roche Applied Scientific) were detected then visualized with Alexa Fluor® 488 or Alexa Fluor® 568.

Mouse MECs were immunostained as previously described¹ . Briefly, MECs were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and then, stained with the following antibodies: anti-E-cadherin (1:50; Sigma) and anti-ZO-1 (5 µg/mL; Life Technologies). Primary antibodies were visualized using secondary antibodies conjugated with Alexa Fluor® 488 or Alexa Fluor® 568 (Life Technologies) and counterstained with DAPI nuclear stain (1 µg/mL; Molecular Probes). Cells were examined using a Leica Inverted Confocal Microscope SP8 (Leica Microsystems, Wetzlar, Germany).