Rabbit anti asc antibody

LPS from Escherichia coli serotype 055:B5 (Toll like receptors 2/4) and nigericin were purchased from Sigma. The recombinant proteins used were human pro-IL-1β, human calmodulin (both from Sino Biological, Philadelphia, PA), and human IL-1β (R&D Systems, Minneapolis, MN). The calcium chelator BAPTA-AM was purchased from Life Technologies, and the calmodulin inhibitors E6 berbamine and W7 were purchased from Enzo Life Sciences (Exeter, UK) and Santa Cruz Biotechnology, respectively. For Western blot analysis, the primary antibodies used were a goat anti-human IL-1β antibody (R&D Systems) or a rabbit anti-human caspase-1 (p10) antibody (Santa Cruz Biotechnology). The secondary antibodies used were a sheep anti-mouse IgG antibody (AbD Serotech, Kidlington, UK) or a goat anti-rabbit antibody (Dako, Copenhagen, Denmark). For immunofluorescence analysis, the primary antibodies used were a rabbit anti-ASC antibody (Santa Cruz Biotechnology), a rabbit anti-calmodulin antibody (Abcam, Cambridge, UK), or a goat anti-human IL-1β antibody (R&D Systems). The secondary antibodies used were an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody or an Alexa Fluor 594-conjugated rabbit anti-goat IgG antibody (both from Life Technologies).

Detection of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) assembly was performed as described previously^{94 (link)}. Briefly, mice were injected with 10⁷ T. gondii ME49-RFP tachyzoites and euthanased 24 h later. Cells were stained for surface molecules, fixed, permeabilized and stained with rabbit anti-ASC antibody (Santa Cruz Biotechnology) for 45 min at room temperature. Subsequently, a secondary anti-rabbit Alexa488 antibody (Life Technologies) was added for 45 min at room temperature. A FMO control without anti-goat Alexa488 was included.
Detection of active caspase-1 by flow cytometry was performed using the carboxyfluorescein FLICA kit (FAM-YVAD-FMK, Immunochemistry Techniques, Bloomington, MN). B6 mice were injected with 10⁷ T. gondii ME49-RFP tachyzoites and 23 h later FAM-YVAD-FMK (diluted in DMSO and PBS) was injected intravenously. Splenic cells were analyzed by FACS 1 h later as described above (24 h after T. gondii ME49-RFP injection). Mice that received T. gondii ME49-RFP but no FAM-YVAD-FMK were used as FMO control.
Single cells were gated for RFP expression and RFP⁺ cells were analyzed for expression of neutrophil, macrophage, monocyte, dendritic cell, T cell and B cell specific surface markers and positivity in green fluorescence as shown in Fig. 4.

To confirmNLRP3 inflammasome formation in HSCs in mouse liver tissue, frozen tissue slides were stained with goat anti-IL-1β antibody (1:100, R&D Systems) and rabbit anti-desmin antibody (1:100, Thermo Fisher) overnight at 4°C after a 30 min blocking with 10% serum and then probed with Alexa 488- or Alexa-555-labeled secondary antibodies (1:500, Invitrogen). Similarly, cells were incubated with goat anti-NLRP3 antibody (1:200, Abcam), rabbit anti-ASC antibody (1:200, Santa Cruz), or rabbit anti-caspase-1 antibody (1:200, Santa Cruz) as we described previously [40 (link), 51 (link)]. Co-localization of IL-1β vs. desmin, NLRP3 vs. ASC or caspase-1 was calculated in Image Pro Plus 6.0 software, and the co-localization coefficient was calculated as the Pearson's correlation coefficient.