Rat anti ly6g

Frozen brain sections or cells were fixed with 4% PFA for 20 min at room temperature and were permeabilized with 1% Triton X‐100 (Solarbio, Beijing, China) solution for 15 min. Next, 5% BSA in PBS with 0.1% triton was used to block non‐specific binding sites at room temperature for 1 h. The slides were incubated with the following corresponding primary antibodies at 4°C overnight: mouse anti‐dsDNA (1:100, Santa Cruz Biotechnology); rabbit anti‐53BP1 (1:5,000, Novus Biologicals); rabbit anti‐Iba1 (1:500, Wako Pure Chemical Industries, Ltd., Japan); rabbit anti‐NeuN (1:500, Abcam); goat anti‐GFAP (1:500, Abcam); rat anti‐Ly6G (1:100, BioLegend); mouse anti‐cGAS (1:100, Santa Cruz Biotechnology); mouse anti‐GSDMD (1:100, Santa Cruz Biotechnology); mouse anti‐caspase‐1 (1:100, Adipogen); and goat anti‐IL‐1β (1:100, R&D Systems). The following day, the sections were washed with cold PBS; the immunoreactions were visualized using fluorescent secondary antibodies. Nuclei were costained with Fluoroshield Mounting Medium containing DAPI (104139, Abcam). All the slides were visualized and photo‐documented using a confocal microscope (Olympus, Heidelberg, Germany) or a Nikon Coolscope digital microscope (Nikon, Tokyo, Japan), and quantified by ImageJ software (NIH).

WT, Trpm2^−/− or mice subjected to neutrophil depletion (anti-Ly6G 1 day before infection and 2 days post-infection) were infected with L. monocytogenes, 72 hpi livers and spleens were dissected, fixed, embedded in paraffin or optimal cutting temperature (OCT) compound and frozen in liquid nitrogen. Sections were cut (4 μm) and stained with rabbit anti-Listeria (Abcam) 1:100, rat anti-Ly6G (Biolegend) 1:100 or rat anti-Ly6C (Santa Cruz Biotechnology) 1:100 plus chicken anti-rabbit IgG Alexa Fluor 488 (Invitrogen) 1:500 and goat anti-rat IgG Alexa Fluor 594 (Invitrogen) 1:500. The slides were prepared with fluoroshield mounting medium with DAPI (Abcam). For the histopathological analysis, paraffin sections of spleens and livers were stained with Hematoxylin and Eosin (H&E) and analyzed by microscopy. The bacterial abscesses were counted in the median lobe of the livers at 72 hpi, whereas in the spleens the severity of the infection was evaluated by comparing the percentage of necrotic follicles.