Rat fitc conjugated anti cd11b antibody

For immunofluorescence staining to visualize lecithinase, BMDMs were stimulated with 60 μg/ml of Alexa Fluor 568 labeled lecithinase for 2 h with or without inhibitors, 50 μM cytochalasin D or 5 mM MCD, 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 h. Cells were incubated with a rat FITC‐conjugated anti‐CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend). For visualization of lecithinase with lysosomes, 4% paraformaldehyde fixed cells were blocked with 10% goat serum with 0.1% saponin in PBS for 1 h. Cells were incubated with a rat LAMP1 antibody (1:500 dilution in 10% goat serum with 0.1% saponin) overnight at 4°C. PBS containing 0.05% Tween‐20 was used to wash between incubation steps. An anti‐rat secondary Alex Fluor 488 antibody (112545143, Jackson ImmunoResearch) was used at 1:500 dilution in 10% goat serum with 0.1% saponin for 1 h at room temperature. The samples were mounted with VECTASHIELD^® Hardset™ Mounting Medium with DAPI (H‐1500, Vector Laboratories, Inc.) and analyzed using either a Leica SP5 confocal microscope or a Zeiss LSM 800 confocal microscope. Z‐stack images of samples were used to generate a three‐dimensional structure using the Imaris Viewer software 9.5.