Anti mouse af546

Cells settled overnight on glass coverslips were washed three times in PBS (pH 7.0) for 5 min after which the cells were fixed in 4% paraformaldehyde for 15 min followed by three washes in PBS as above. The cells were permeabilized in permeabilization buffer (PBS containing 5% goat serum, 10% sucrose and 1% Triton X-100) for 15 min. The cells were incubated with anti-p40 [6 (link)] (biotin-linked antibody 1:1000), anti-dsRNA (clone K1 mouse 1:1000; Product Number 10020500 English & Scientific Consulting) and anti-CP anti-serum [6 (link)] (rabbit 1:1000) in permeabilization buffer for 90 min then incubated with 20 μg mL^-1 anti-biotin AF488 (Invitrogen; S11223), anti-mouse AF546 (Invitrogen; A11003) and anti-rabbit AF633 (Invitrogen; A21070) in permeabilization buffer for 30 min. Cells were washed thrice, for 15 mins. In the second wash, 1 μg mL^-1 DAPI was added to stain the nuclei of the cells. Cells were mounted using fluorescent mounting medium (DAKO, S3023). Cells were imaged using a Zeiss LSM780 laser scanning confocal microscope using the x63, 0.75 NA objective. All images were acquired using the same exposure and detector settings for each spectral channel. The Zen 2011 Blue software was used to acquire images from the Zeiss LSM780 microscope and to perform image overlays.