Alexafluor 594 donkey anti sheep

Cells were plated in 35-mm glass bottom plates from MatTek Corporation (Ashland, MA). After treatment, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 15 min. For Z-DNA staining, a 4% paraformaldehyde solution containing 0.1% Triton-X100 in PBS was added to cells for 15 min immediately after removal of media. Cells were then washed three times with PBS. Blocking was done in 3% BSA, 0.1% Triton-X100 in PBS. Primary antibodies: Z-DNA from Abcam (Cambridge, UK, cat# ab2079) was used at 1:200 dilution. dsRNA antibody (J2) from Scicons (Hungary) was used at 1:50 dilution. AlexaFluor 488 or 594 donkey anti-mouse (Invitrogen, cat# A21206; 1:1000) and AlexaFluor 594 donkey anti-sheep (Jackson ImmunoResearch, West Groove, PA, cat# 713-585-147; 1:500) were used as secondary antibodies. Antibodies were diluted in 0.5% BSA +0.05% Triton X100 in PBS. After each antibody incubation, cells were washed three times with 0.05% Triton X100 in PBS. For DNA counterstaining, 1 µg/ml solution of Hoechst 33342 in PBS was used. Images were obtained with a Zeiss Axio Observer A1 inverted microscope with N-Achroplan 100×/1.25 oil lens, Zeiss MRC5 camera, and AxioVision Rel.4.8 software.

Cells were plated in 35 mm glass bottom plates from MatTek Corporation (Ashland, MA, USA). After treatment, cells were washed with 1× PBS and fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were then washed three times with 1× PBS. Blocking was completed in 3% BSA, 0.1% Triton-X100 in 1× PBS. Anti-interferon alpha (Abcam, Cat# ab196221) and Anti-IL-28A (Abcam, Cat# ab233754) antibodies were used to detect IFN-α and IFN-λ proteins, respectively. AlexaFluor 488 donkey anti-mouse (Invitrogen, Cat# A21206) and AlexaFluor 594 donkey anti-sheep (Jackson ImmunoResearch, cat# 713-585-147) were used as secondary antibodies. Antibodies were diluted in 0.5% BSA + 0.05% Triton X100 in 1× PBS. After each antibody incubation, cells were washed three times with 0.05% Triton X100 in 1× PBS. For DNA counterstaining, a 1 µg/mL solution of Hoechst 33342 (Cat# H1399, Sigma-Aldrich, St. Louis, MO, USA) in 1× PBS was used. Immunofluorescence images were acquired with a 100× oil-immersion lens using the Zeiss confocal microscope (LSM700, Opti-Ups 1000B).