Immunofluorescence Staining of TREM2 and GSDMD

Cells were seeded into 8-well LabTek removable chamber slides (Thermo Scientific, 177402). After treatment, cells were fixed with 4% paraformaldehyde (PFA) for 15 min before permeabilization with 0.1% saponin in blocking buffer (1% fatty acid free BSA, 20 mM glycine in PBS) for 15 min at room temperature. For non-permeabilized TREM2 staining, saponin was omitted from all blocking and washing steps. Then, samples were incubated with primary antibodies (rabbit polyclonal anti-TREM2, Proteintech, 13483–1-AP, 1:100, overnight at 4 °C; rabbit polyclonal anti-GSDMD, Abbexa, abx340202, 1:250, 3 h at 37 °C), followed by 1-h incubation at room temperature with the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Sci., A-11008, 1:400). From each well, 3 non-overlapping images from the top, middle and bottom areas were randomly taken using a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems) with a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit.