Rabbit anti giantin

Fibroblasts from the affected individual A‐II‐2 and appropriated controls were seeded on coverslips. The day after they were incubated with 5 μg/mL Brefeldin A (BFA) for 9 minutes and immediately fixed in 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed three times in phosphate buffered saline (PBS) and afterward permeabilized and blocked with 0.1% saponin in 3% BSA for 20 minutes at room temperature. Immunofluorescence staining was performed for the Golgi markers GM130 (mouse anti‐GM130; BD Transduction Laboratories) and giantin (rabbit anti‐giantin; Covance) overnight in 3% BSA in 1x PBS. Cells were washed three times in PBS and secondary antibodies anti‐mouse IgG Alexa Fluor 555 (Invitrogen, Molecular Probes) and an anti‐rabbit IgG Alexa Fluor 488 (Invitrogen, Molecular Probes) were applied. DNA was stained by DAPI and cells were mounted in Fluoromount G (SouthernBiotech). Pictures were taken with a fluorescence microscope (BX60, Olympus). At least 100 cells per sample were counted and the experiment has been performed two times. Significance levels were calculated using Student's t‐test.

HeLa cells were seeded onto 12 mm glass coverslips 24 h before transfection of the appropriate construct, followed by infection with C. trachomatis serovar L2. 16–18 h post-infection, cells were fixed in 4% paraformaldehyde (Acros Organics/Thermo Scientific; Logan, UT) and permeabilized with 0.5% TritonX-100 for 5 min. Coverslips were incubated with primary antibody: mouse anti-FLAG M2 (Sigma Aldrich), rabbit anti-IncA (Ted Hackstadt, Rocky Mountain Laboratories, Hamilton, MT), rabbit anti-IncG (Ted Hackstadt), or rabbit anti-giantin (Covance; Emeryville, CA), followed by incubation with the appropriate secondary antibody conjugated to DyLight fluors (Jackson ImmunoResearch Laboratories; West Grove, PA). Coverslips were mounted on glass microscope slides using Prolong Gold mounting reagent (Life Technologies). Slides were visualized with an 60X objective and 2x zoom using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope.