Polyclonal rabbit anti human c3c complement

Purified EBs from C. trachomatis D and L2 were incubated with an equal volume of either NHS or HIHS for 30 minutes at 37 °C. EBs were washed twice in PBS with centrifugation at 20000 x g for 15 minutes between each wash. Samples were boiled in RunBlue LDS Sample Buffer (Expedeon, CA, USA) containing 5% v/v β-mercaptoethanol and proteins were separated on a 7,5% SDS polyacrylamide gel according to Laemmli (Laemmli 1970). Proteins were blotted on a nitrocellulose membrane according to Drasbek et al. (Drasbek 2004 ). The membrane was blocked in Tris buffered saline (TBS) with 3% gelatin. Polyclonal Rabbit Anti-Human C3c Complement (Agilent Technologies) (1:1000) was used as primary antibody and Anti-Rabbit IgG Alkaline Phosphatase (Sigma-Aldrich) (1:20,000) was used as secondary antibody. Protein bands were developed by adding BCIP/NBT alkaline phosphatase substrate (Kem-En-Tec Diagnostics, Taastrup, Denmark).

The following primary antibodies were used in this study: Anti-human CD11b (MEM-174) (ImmunoTools GmbH, Friesoythe, Germany), Polyclonal Rabbit Anti-Human C3c Complement (Agilent Technologies, Glostrup, Denmark), Mab32.3 against C. trachomatis MOMP [16] , and PAb17 against C. trachomatis outer membrane [17] . FITC-, Alexa Flour® 488-, and rhodamine-conjugated secondary antibodies were purchased from Jackson ImmunoResarch (Jackson ImmunoResearch, PA, USA). Anti-Rabbit IgG Alkaline Phosphatase was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Goat anti-rabbit antibody conjugated with 10 nm colloidal gold (British BioCell, Cardiff, UK) was used for immunoelectron microscopy.