Rabbit anti asc

For DC immunostaining, DCs generated using FLT3L were cultured on coverslips and either left untreated (none) or treated with LPS alone for 18 hours, or BMDCs were primed with LPS for 3h then treated with PGPC or Alum for 15h. Cells were washed then fixed with 4% PFA for 15 minutes at room temperature, and blocked using 1% goat serum for 1 hour. For immunofluorescence staining of hyperactive DCs that migrated to the dLN, cells were sorted from the dLN as CD11c⁺ CD45.2+ CD45.1⁻ CFSE⁺ live cells and plated onto glass 96 well plates. DCs were stained with iFluor 488 phalloidin (ThermoFisher) and DAPI. For anti-ASC speck staining, hyperactive DCs were sorted from the dLN as CD11c⁺ CD45.2+ CD45.1^neg and resident DCs as CD11c⁺ CD45.2^neg CD45.1⁺ then plated on glass 96 wells flat plate. Cells were left to rest in media for 3–4hours, then fixed using 4% PFA. DCs were stained with Alexa Fluor 647 conjugated Phalloidin (ThermoFisher), rabbit anti-ASC (Adipogen) followed by Alexa Fluor 488-conjugated chicken anti-rabbit IgG (ThermoFisher). ASC-citrine hyperactive DCs or resident DCs were sorted from the dLN then fixed with 4% PFA for 15 minutes at room temperature, blocked using 1% goat serum for 1 hour, then stained with Alexa Fluor 647 conjugated Phalloidin and DAPI. Images were acquired using 20x or 63x oil immersion lenses on Zeiss microscope.