A 11030

After the dissected brain tissues were fixed in 4% formaldehyde at 4 °C overnight, they were washed with 0.1 M PBS twice for 15 min each (pH 7.4). Thick sections of the brains (30 μm) were cut with a vibrating blade microtome (VT 1200 S, Leica, Wetzlar, Germany), washed in 0.1 M PBS for 15 min, and then incubated in 0.1 M PBS containing 5% normal goat serum (NGS, Boster, China) for 1 h at room temperature. The primary anti-Dop1 and anti-AC2 (custom made, see “Protein preparation and Western blot analysis” section of Methods for details) was diluted at 1:300 in 0.1 M PBS containing 2% NGS. Incubation with primary antibodies lasted for 48 h. The tissues were washed with 0.1 M PBS three times for 15 min each and subsequently incubated with the mixture of two secondary antibodies, Goat anti-rabbit antibody Alexa fluor 488 (1: 500, A11034, Life Technology) and Goat anti-mouse antibody Alexa fluor 546 (1: 500, A11030, Life Technology), for 1 h at room temperature. After washing three times, the tissues were mounted in anti-fade fluorescence mounting medium. The negative serum of Dop1 and AC2 from rabbit and mouse were used as the negative control. The nucleus of locust brain is labeled by Hoechst33342 (Life Technology) to indicate the brain structure. The fluorescence was detected using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany).

Mock- or C. burnetii-infected WT BMDM and STING^gt/gt were seeded onto coverslips in 8-well iBIDI chamber slides at a confluency of ~9 × 10⁴ cells/well. At 12 dpi cells were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.1% Triton X-100 for 15 min at room temperature and blocked in 2% FBS in TBS for 45 min at 37 °C. Primary antibody labeling was completed with anti-STING (1:50), anti-IRF3 (1:100) and anti-BAX (1:100) overnight in a humified chamber at 4 °C. Secondary antibody labeling was completed using anti-rabbit (Life Technologies A11034 or A11035) or anti-mouse (Life Technologies A11029 or A11030) Alexa Fluor 488 or 546 (1:300) by incubating membranes for 1 h at room temperature in the dark. Samples were stained with DAPI (1:100; Cell Signaling 4083), mounted onto coverslips using ProLong Diamond Antifade Mount (Invitrogen P36961), and imaged using a Leica DMi8 fluorescence microscope. For mitochondrial staining 100 nM MitoTracker Red CMXRos (Invitrogen M7512) was treated to mock or infected BMDMs and incubated at 37 °C in 5% CO₂ for 15 min followed by washing with incomplete DMEM. After staining with Mitotracker dye cells were fixed with protocol used above. Leica Application Suite X Imaging and Analysis Software was used to process the final images.

Secondary antibodies used for immunohistochemistry were goat anti-mouse (IgG 488, Invitrogen, A11029, 1:1,000), goat anti-rabbit (IgG 488, Invitrogen, A11034, 1:1,000), goat anti-rabbit (IgG 546, Invitrogen, A11035, 1:1,000), goat anti-mouse (IgG 546, Invitrogen, A11030, 1:,1000), goat anti-rat (IgG 647, Life Technologies, A21247, 1:1,000).

The whole procedure was carried out at room temperature. Transfected HEK cells were fixed with PBS + 4% paraformaldehyde for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH4Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (Sigma, #S7900) for 5 min, washed once (5 min) with PBS + 0.2% BSA (PBS-BSA), and incubated for 30 min with the antibody-containing supernatants. After 3 washes (5 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary anti-mouse or anti-human or anti-rabbit IgG conjugated to AlexaFluor 546, 647 or 488 (A-11030, A-21236, A-11029, A-21445, A-21245, 1:400, Life Technologies). After 3 washes (5 min) with PBS-BSA, cells were mounted in Möwiol (Fluka, #33480). Pictures were taken using a Zeiss LSM700/800 confocal microscope, with a 63x oil immersion objective.

Cells were fixed for 20 minutes with 4% paraformaldehyde-PBS, permeabilized with 0.5% Triton X-100 (#X100, Sigma-Aldrich) for 15 minutes and incubated with 5% BSA (Bovine Serum Albumin) in PBS for 1 hour at room temperature. After saturation, cells were incubated overnight at 4°C with anti-TMS1/ASC antibody (ASC) (#ab155970 rabbit, Abcam, 1/200 dilution) or anti-spike (S) antibody (#GTX632604 mouse, Genetex, 1/100 dilution) in PBS-BSA 5%. Then, cells were incubated with appropriate secondary antibody conjugated to Alexa Fluor 488 (#A11034, Invitrogen, 1/500) or Alexa Fluor 546 (#A11030, Invitrogen, 1/500) at room temperature for 1 hour. Nuclei were stained with Hoechst 33342 (#1874027, Invitrogen, 1/1000 dilution) for 30 minutes at room temperature. After three PBS washes, cells were mounted with Fluoromount G medium (Southern biotech). Cells were analyzed and imaged by Leica Dmi8 microscope using a 60X objective.