Asc tms1

Macrophages were fixed for 2 h with 4% paraformaldehyde in PBS at 24 h post‐infection. Cells were then washed three times with PBS and permeabilized for 15 min with 2% BSA, 2% saponine, 0.1% Triton x‐100 in PBS at RT. After three washes with PBS, samples were blocked for 1 h with 2% BSA in PBS at RT. Rabbit primary antibodies anti‐cleaved Caspase‐8 (Cell Signaling), ASC/TMS1 (Cell Signaling), cleaved Caspase‐1 (Cell Signaling), phospho‐RIP3 (Cell Signaling), phospho‐MLKL (Cell Signaling), mouse CD45.1‐AlexaFluor647 (BioLegend) were diluted 1:300 in 2% BSA in PBS and used for primary staining of the cells overnight at 4°C. After three washes in 2% BSA in PBS, samples (excluding those stained with anti‐mouse CD45.1‐AlexaFluor647) were incubated with a secondary goat anti‐rabbit antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific) diluted 1:300 in 2% BSA in PBS for 1 h at RT. Samples were stained with 1:1,000 Hoechst (ThermoFisher Scientific) and stored in PBS at 4°C before imaging by fluorescence microscopy.