Dc 350 fx camera

Synchronized adult worms were transferred to glass slides in 15 μl of M9 solution. Samples were permeabilized, fixed and washed with absolute ethanol, then 15 μl of a 2 ng/μl solution of 4′, 6′-diamidino-2-phenylindole hydrochloride (DAPI) diluted in M9 was added. Quantitative analysis was performed on z series of images acquired using a Leica DM6000 fluorescence microscope, Leica DC 350 FX camera under the control of Leica LAS AF 6000 software. Optical sections were collected at 0.50 μm increments.

Producer or infected cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, France) for 15 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 7 min. Fixed cells were then saturated with 3% bovine serum albumin (BSA)/PBS for 20 min and incubated for 1 h with primary antibodies diluted in 1% BSA/PBS at the following dilutions:^{80 (link)} anti-HDAg SE1679 rabbit polyclonal serum, 1/500; anti-DENV-E 3H5 mAb, 1/800; anti-NS5A 9E10 mAb, 1/1,000; and anti-HBcAg serum, 1/500. After three washes with 1% BSA/PBS, cells were incubated for 1 h with the corresponding secondary antibodies (Molecular Probes, The Netherlands) at a 1/1000 dilution: donkey anti-rabbit Alexa Fluor 488 (Cat # A-21206); donkey anti-mouse Alexa Fluor 555 (Cat # A-31570); and goat anti-human Alexa Fluor 555 (Cat # A-21433) sera. Cells were washed three times with PBS and then stained for nuclei with Hoechst 33342 (Molecular Probes) for 5 min. After two washes in PBS, cells were imaged with an Axiovert 135 M microscope (Zeiss, Germany) equipped with a DC350FX camera (Leica, Germany), and images were analyzed with the ImageJ software (imagej.nih.gov).