Ultraview spinning disk confocal microscope

To stain proteins for immunofluorescence microscopy, cells were fixed with 4% formaldehyde and permeabilized in permeabilization buffer (PS; 0.05% saponin, 1% BSA, and 0.05% NaN₃ in PBS) for 20 min. Samples were incubated with rabbit anti-ASC (1:200 in PS) for 2 h, washed with PBS, and subsequently incubated with Alexa Fluor 594–coupled goat anti–rabbit IgG (1:1,000), Hoechst 33342 (1:5,000), and, where indicated, with Alexa Fluor 647–coupled VHH_ASC or VHH NP1 (1 µg/ml) for 1 h. Samples were washed with PBS and H₂O, and mounted with Fluoromount-G (Southern Biotech) or Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich). Images were acquired using a PerkinElmer Ultraview Spinning Disk Confocal microscope or a Nikon Eclipse Ti wide field microscope. ASC foci and nuclei in wide field microscopy images were quantified using the spot detection function of the Imaris software package (Bitplane).

All imaging experiments were carried out with a PerkinElmer Ultraview Spinning Disk confocal microscope equipped with a Nikon Apochromat TIRF 100X 1.49NA objective and a Hamamatasu C9100-23B EMCCD camera (PerkinElmer). Images were acquired at room temperature with log-phase cells sandwiched between an EMM5S agarose pad and a coverslip. For high-temporal resolution imaging, stack images containing three planes with 0.5 μm spacing were taken every 5 sec by Volocity (PerkinElmer) unless otherwise specified; for single time-point imaging, stack images containing 11 planes with 0.5 μm spacing were acquired.