After washing, HRP conjugated goat anti human IgG in combination with substrate ABTS was used to visualize the primary antibody bind ing. selleck chemicals llc The human antibody binding to chlamydial fusion proteins was quantitated by reading the absorbance at 405 nm in a microplate reader. In some assays, the human antibody samples were further absorbed with lysates Inhibitors,Modulators,Libraries made from either HeLa cells alone or C. trachomatis serovar D infected HeLa cells at 4 C overnight in addition to the bacterial lysate absorp tion. 3. Immunofluorescence Inhibitors,Modulators,Libraries assay HeLa cells grown on coverslips were fixed with 2% para formaldehyde dissolved in PBS for 30 min at room temperature, followed by permeabiliza tion with 1% saponin for an additional 30 min. After washing and blocking, the cell samples were sub jected to antibody and chemical staining.

Hoechst was used to visualize nuclear DNA. A rabbit anti chlamydial organism antibody plus a goat anti rabbit IgG secondary antibody conjugated with Cy2 was used to visualize chlamydial inclusions. The various mouse antibodies plus a goat anti mouse IgG conjugated with Cy3 were used to visualize the corresponding antigens. The mouse antibodies include pAbs made against Inhibitors,Modulators,Libraries the pORF5 GST fusion proteins and mAbs 100a against CPAFct C terminus, MC22 against the major outer membrane protein and BC7. 1 against chlamydial HSP60. In some cases, the pri mary antibodies were human Inhibitors,Modulators,Libraries IgG molecules purified with the corresponding GST fusion proteins conjugated to glu tathione agarose beads. To visualize the binding of the purified human IgG antibodies to chlamydial antigens, a goat anti human IgG conjugated with Cy3 was used.

The cell samples after the appropriate immuno labeling were used for image Inhibitors,Modulators,Libraries analysis and acquisition with an Olympus AX 70 fluorescence microscope equipped with multiple filter sets Deltarasin? as described previously. Briefly, the multi color labeled sam ples were exposed under a given filter set at a time and the single color images were acquired using a Hamamatsu digital camera. The single color images were then super imposed with the software SimplePCI to display multi colors. An Olympus FluoView Laser Confocal Micro scope was used to further analyze the co stained samples at the UTHSCSA institutional core facility as described previously. All microscopic images were processed using the Adobe Photoshop program. 4. Western blot assay The Western blot assay was carried out as described else where. Briefly, the purified fusion protein, Chlamydia infected cell cytosolic fraction or antibody precipitated endogenous chlamydial protein samples were solublized in 2% SDS sample buffer and loaded into SDS polyacrylamide gel wells. The cytosolic fraction also called S100 was prepared as previously described.