Anti mouse secondary rhodamine red antibody

For visualization of inflammasomes, untreated or treated BMDMs were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 10% normal goat serum (005000121, Jackson ImmunoResearch) supplemented with 0.1% saponin (47036, Sigma) for 1 hr. Cells were incubated with a rabbit anti-ASC antibody (1:500 dilution, clone AL177, AG-25B-0006-C100, AdipoGen) overnight at 4 °C. An anti-rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) was used. Cells were counterstained in DAPI mounting medium (H-1200, Vecta Labs). Inflammasome specks and BMDMs were visualized, counted, and imaged using a Leica SP5 confocal microscope. For visualization of HBL and cell membrane, BMDMs were stimulated with HBL component B (5 μg/ml) for 1 hr, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 hr. Cells were incubated with a mouse anti-HBL-B antibody (1:200 dilution in 1% BSA)⁴⁰ and a rat FITC-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 °C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) was used. BMDMs were analysed using a Leica SP5 confocal microscope.