Plan apochromatic 60 na1.42 objective

LX-2 cells were infected with B. abortus, and after 24 h, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, and blocked with PBS containing 1% BSA for 1 h. Infected cells were stained with mouse anti-ASC (Santa Cruz Biotechnology) diluted in 0.1% PBS–Tween 20 overnight at 4°C. Cells then were incubated with rabbit anti-mouse Alexa Fluor 488 (Molecular Probes, Life Technologies) diluted in 0.1% PBS–Tween for 4 h at room temperature. 4,6-Diamidine-2-phenylindole (DAPI) was used for nuclear staining, and cells were stained for 30 min at room temperature. After washing in PBS, cells were mounted and then analyzed by fluorescence microscopy. Confocal images were analyzed using FV-1000 confocal microscope with an oil immersion Plan Apochromatic 60 × NA1.42 objective (Olympus).

THP-1 cells (2 × 10⁵ cells/well) were incubated in chamber-slides with 10 ng of PMA/ml for 24 h to promote adherence. Then, cells were treated as indicated in each figure, fixed with 2% paraformaldehyde and permeabilized with 0.1% saponin. The samples were then incubated with an anti-HLA-ABC class I mAb (W6/32) followed by Alexa 633-labeled secondary Ab (Molecular Probes Life Technologies) and an anti-CD61 Ab (VI-PL2; BD Bioscience) followed by an Alexa 546-labeled secondary Ab (Molecular Probes Life Technologies).
In all cases, slides were mounted with PolyMount (Polysciences) and analyzed using a FV-1000 confocal microscope with an oil-immersion Plan Apochromatic 60× NA1.42 objective (Olympus). The obtained images were processed with FIJI software (open source).