Goat anti rat igg

The testis, ovary, and hypothalamus of zebrafish were fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated in graded alcohols, cleared in toluene, and embedded in paraffin wax. Serial transverse sections 5 µm thick were dried at 60°C for 2 h and placed on glycerin/albumin-coated slides. Dewaxed sections were rehydrated and washed in phosphate buffered solution (PBS), then incubated with PBS containing 3% hydrogen peroxide for 30 min to quench endogenous peroxidase activity. Next, the sections were incubated with Gαq antibody (ABclonal, China) overnight at 4°C. For the negative control, normal pre-immune rabbit serum was used to replace the Gαq antibody. The sections were then washed thrice with PBST and incubated with goat-anti-rat IgG (Abcam, Britain 1:5,000) at 37°C for 45 min. Following this, DAB chromogenic solution (Maxim, China) was used for 3–10 min, and hematoxylin (BaSO, China) was applied for 1 min to stain the nucleus. The sections were photographed by an Olympus microscope (Olympus, Japan). The protein sample of the zebrafish brain tissue was prepared as described before (Xiong et al., 2017 (link)). Western blot assay was then applied to verify the specificity of the commercial Gαq antibody. The detailed WB protocol was referred to in a previous study (Xiong et al., 2017 (link)).

The lung, liver and kidney tissue samples from Leptospira-infected mice mentioned above were fixed with 4% formalin and then embedded and sectioned. CD11b or Ly6G has been considered as mouse peripheral blood-derived mononuclear-macrophage or neutrophil surface makers, respectively [41 (link),42 (link)]. Using 1:100 diluted rabbit anti-mouse CD11b or rat anti-mouse Ly6G-IgG as the primary antibody (Abcam) and 1:1000 diluted HRP-conjugated goat anti-rabbit-IgG or goat anti-rat-IgG as the secondary antibody (Abcam), an immunohistochemical method was used to detect the mononuclear-macrophages and neutrophils in the tissues from peripheral blood. The stained mononuclear-macrophages and neutrophils (brown) per mm² section were quantitatively estimated using Image-Pro Plus software (Nikon, Japan) [43 (link)–45 (link)]. In the detection, the mice injected with EMJH liquid medium were used as the control. In addition, the efficiencies of the CD11b-IgG and Ly6G-IgG were determined using M-CSF-induced Ms-macrophages and Ms-neutrophils from peripheral blood before use.

Antibodies used for western bloting included F4/80 (rat monoclonal, Abcam, ab16911; 1:200), CD11b (rabbit monoclonal, Abcam, ab133357; 1:1000), LY6C (rat monoclonal, Novus, NBP2-00441; 1:1000), MCP-1 (rabbit polyclonal, Cell Signaling Technology, #2029; 1:1000), NLRP3 (rabbit monoclonal, Cell Signaling Technology, #15101; 1:1000), pro-Caspase1 (rabbit monoclonal, Abcam, ab179515; 1:1000), GSDMD (rabbit monoclonal, Abcam, ab209845; 1:1000), IKK (rabbit monoclonal, Abcam, ab178870; 1:1000), phospho-IKK (rabbit monoclonal, Cell Signaling Technology, #2697; 1:1000), p65 (mouse monoclonal, Sabta cruz, sc-8008; 1:200), phospho-p65 (rabbit monoclonal, Cell Signaling Technology, #3033; 1:1000), IĸBα (mouse monoclonal, Cell Signaling Technology, #9246; 1:1000), phospho-IĸBα (mouse monoclonal, Cell Signaling Technology, #4814; 1:1000), GAPDH (mouse monoclonal, Abcam, ab8245; 1:8000), Goat Anti-Mouse IgG (Abcam, ab97023; 1:10000), Goat Anti-Rabbit IgG (Abcam, ab97051; 1:10000) and Goat Anti-Rat IgG (Abcam, ab97057; 1:10000). Antibodies used for immunohistochemistry and immunohistochemistry-frozen included F4/80 (rat monoclonal, Abcam, ab16911; 1:50) and LY6C (rat monoclonal, Novus, NBP2-00441; 1:200). LPS, IFN-γ, IL-4, IL-10 and M-CSF were purchased from PeproTech, ATP and nigericin were purchased from Med Chem Express.

10⁵ isolated human or mouse neutrophils were seeded on glass coverslips in tissue-culture plates and incubated with PMA (100 ng/ml, Sigma-Aldrich), Ionomycin (1 µM, Sigma-Aldrich), or 5 × 10³C. albicans preformed hyphae for 2.5 h at 37°C. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.01% TritonX-100 for 10 min, and blocked with 5% donkey serum for 30 min at room temperature. Cells were stained with goat polyclonal anti-MPO (R&D systems), rabbit polyclonal anti-CitH3 (Abcam), and combined rat anti-S100A8 and anti-S100A9 (clone 10 and clone 46 respectively, kindly provided by C. Urban, Umeå, Sweden). Secondary antibodies were donkey anti-goat IgG (Jackson ImmunoResearch), goat anti-rat IgG (Abcam), and goat anti-rabbit IgG (Jackson ImmunoResearch). The PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was used to stain lipids of the neutrophil plasma membranes. DNA was stained with 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and NETs were visualized using a Leica SP8 inverse confocal microscope and analyzed with FIJI software.