Fluoview fv1000 d confocal laser scanning microscope

Immunofluorescence double-staining was performed to confirm the intracytoplasmic localization of PG or TF in epithelial cells. According to the method described previously^{41 (link)}, tissue sections were first reacted with the anti-PG or anti-TF antibody using the avidin-biotin-complex method with fluorescein isothiocyanate-conjugated streptavidin (DAKO), and microwaved again for 20 min. After these procedures, the sections underwent the secondary reaction with anti-human cytokeratin AE1/AE3 antibody (clones:AE1/AE3; DAKO) diluted 1:50, followed by anti-mouse immunoglobulin conjugated with tetra-methylrhodamine isothiocyanate (DAKO). Stained sections were observed and photographed using a FLUOVIEW FV1000-D confocal laser scanning microscope (FV10-ASW1.6 software version; Olympus Co.). Samples showing intracellular positivity were used to acquire consecutive cross-sectional images (XYZ) using the line sequential scanning version of the same microscope. The acquired images were used to produce images in the XY plane, and to confirm the intracellular localization of bacteria, orthogonal projections were produced in the XZ and YZ planes.

LabTek^TM chambers were precoated with 2 μg/mL clustered Fc, ephrin-B2/Fc or Eph-B4/Fc at 4°C overnight, followed by incubation with 2 × 10⁴ cells at 37°C for 4 h. After washing with PBS, adherent cells were fixed with 4% (w/v) paraformaldehyde for 20 min and permeabilized with 0.1% (v/v) Triton X-100 for 10 min. Actin filaments were stained with rhodamine-phalloidin (1:1000, Invitrogen) for 45 min. After washing with PBS, the chambers were mounted using an antifade mounting fluid containing DAPI (4',6-diamidino-2-phenylindole), and images were captured using a Fluoview FV-1000D confocal laser scanning microscope (Olympus, Tokyo, Japan).