Lsm980

Worms were anesthetized using 100 mM sodium azide (NaN₃) and mounted on 5% agar on glass slides. Worms were analyzed by Nomarski optics and fluorescence microscopy, using a ×40 or ×63 objective on a confocal laser-scanning microscope (Zeiss LSM880 and LSM980) or a spinning disk confocal (Nikon W1). When using GFP, we estimated the resolution of our confocal to be ~250 nm. 3D image Z-stacks were converted to maximum intensity projections using Zeiss Zen Blue or ImageJ software (Schindelin et al., 2012 (link)). Manual quantification of puncta was performed by scanning the original full Z-stack for distinct dots in the area where the processes of the two neurons overlap. Figures were prepared using Adobe Illustrator.

Cells were seeded in a 96-well plate at a density of 5 × 103 cells/well. First, the cells were treated with different concentrations of ibuprofen (C643 at 0.1.5 mM and OCUT at 0.3 mM) for 48 h. Next, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100. Then, the cells were incubated with the primary antibody at 4 °C overnight, and then the secondary antibody was added and incubated for 1 h. Immunofluorescence was performed with the following antibodies: rabbit anti‐ASC (Servicebio, GB115270), rabbit anti‐NLRP3 (SAB, 29125), rabbit anti‐GSDMD (Proteintech, 20770-1-AP), goat Anti-Rabbit IgG (abbkine, A23220) for Dylight 488, and Goat Anti-Rabbit IgG (abbkine,A23620) for Dylight 649. DAPI (Solarbio, S2110) was used as a nuclear counterstain. The images were observed with a Nikon Ti Eclipse Confocal Microscope and an LSM 980 using basic operating techniques.

For most of the Z-Stack images taken on the Zeiss LSM980 with AiryScan or Nikon N-SIM S microscopes and processed in ImageJ, each image was loaded into ImageJ and brightness and contrast were modulated solely by using the reset command (autoscale) on the brightness and contrast panel. This command restores the original brightness and contrast settings, setting the display range to the full pixel value of the image, and rendering processed images as similar as possible to native images. For certain images (listed in the relevant figures), the intensity values for each channel were matched to those of a previously “autoscaled” parasite using the SET command on FIJI.