Multiple filter sets

HeLa cells grown on coverslips were fixed with 4% paraformaldehyde (Sigma) dissolved in PBS for 20 min at room temperature followed by permeabilization with 0.1% Triton X-100 (Sigma) for an additional 10 min. After washing and blocking, the cell samples were subjected to antibody and chemical staining. DAPI (blue; Sigma) was used to visualize nuclear DNA. A rabbit anti-chlamydial organism antibody (R12AR39, raised with C. pneumoniae AR39 organisms; unpublished data) or, in some cases, a rabbit polyclonal antibody against IncA (kindly provided by Dr. Ted Hackstadt, ref: [8 (link)], plus a goat anti-rabbit IgG secondary antibody conjugated with Cy2 (Jackson ImmunoResearch Laboratories) was used to visualize chlamydial inclusions. Mouse monoclonal antibodies raised against GSK3α/β (BD laboratories) or β-catenin (BD laboratories) plus a goat anti-mouse IgG conjugated with Cy3 (red, Jackson ImmunoResearch) were used to visualize the corresponding antigens. For the transfected cell samples, the RFP-fusion proteins were visualized via the fusion tag RFP (red). The images were acquired with an Olympus AX-70 fluorescence microscope equipped with multiple filter sets (Olympus) as described previously [36 (link),37 (link)]. The single color images were superimposed with the software SimplePCI. All microscopic images were processed using Adobe Photoshop (Adobe Systems).

HeLa cells grown on coverslips with chlamydial infection were fixed and permeabilized for immunostaining as described previously [16] (link), [17] (link). Hoechst dye (blue, Sigma) was used to visualize nuclear DNA. For titrating IFUs from swab and tissue homogenate samples, a mouse anti-chlamydial LPS antibody (clone# MB5H9, unpublished observation) plus a goat anti-mouse IgG conjugated with Cy3 (red; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used to visualize chlamydial inclusions. All immunofluorescence-labeled samples were observed under an Olympus AX-70 fluorescence microscope equipped with multiple filter sets (Olympus, Melville, NY).