Ab184308

Paraffin section of brain tissue was obtained after transcardial perfusion and primary cortical microglia was fixed by 4% paraformaldehyde after LPS + ATP treatment, then followed by overnight incubation with primary antibodies: primary anti-Cd11b antibodies (ab184308, Abcam, Cambridge, UK), anti-Gfap (#80788, CST, MA, USA), anti-Nlrp3 antibodies (19771-1-AP, Proteintech, Wuhan, China), anti-caspase1 antibodies (22915-1-AP, Proteintech), anti-caspase6 antibodies (ab185645, Abcam), anti-Il-1β (#12242, CST), and anti-α-tubulin (#3873, CST), and then incubated with secondary antibodies. Cell nuclei were stained with DAPI, 6 random high-power fields were chosen and images were obtained using a fluorescence microscope (Leica, Oskar-Barnack, Germany).

After μCT scanning, 6 femur specimens per group were fixed in 4% paraformaldehyde for at least 48 h, and washed three times with PBS buffer. Samples were decalcified in 10% EDTA (E1171, Solarbio, China) on a shaker at 4°C, the solution was changed every 3 days for 21 days. The specimens were then washed 3 times with PBS buffer and subjected to dehydrated, paraffin-embedded, and sectioned 4 μm slides. HE staining was performed in order to analyze the inflammation and general changes of bone microstructure at histological and cytological levels.
Immunofluorescence staining was applied to analyze MDSCs polarization to M1 according to standard protocols. The sections were incubated at 4°C overnight with primary antibodies rabbit anti-mouse CD11b (ab184308, 1:500, Abcam, United States) and rabbit anti-mouse CXCL10 (10H11L3, 1:500, Thermo Fisher, United States) for multiplex, respectively; the corresponding secondary antibodies were added onto the sections for 1 h. For immunofluorescence, slides were counterstained with DAPI. The slide images were observed and captured by Eclipse Ti-SR microscope (Nikon, Japan). ImageJ was used for the quantitative analysis if necessary.

Livers were fixed with 4% formalin for 24 h and subsequently embedded in paraffin and cut into 5-μm-thick sections. For liver histopathology, samples were stained with hematoxylin and eosin (H&E). For immunohistochemical (IHC) staining, samples were dehydrated, exposed to antigen, and then incubated with IL-1β (ab283818, Abcam, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), and Ly-6G (ab261916, Abcam, 1:100 dilution) antibodies respectively at 4 °C overnight. For immune-fluorescence (IF) staining, tissue sections or cultured cells were fixed with 4% formalin for 30 min and then incubated at 4 °C overnight with antibodies against CD11b (ab184308, Abcam, 1:100 dilution), CD68 (#26042, Cell signaling Technology, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), NR4A1 (ab153914, Abcam, 1:100 dilution); SOX9 (ab185966, Abcam, 1:100 dilution), NEK7 (sc-393539, Santa Cruz Biotechnology, 1:100 dilution), and NLRP3 (ab270449, Abcam, 1:100 dilution). Then samples were incubated with the secondary antibody conjugated to Alexa Fluor 488 (Jackson Immunoresearch) or Alexa Fluor Cy5 (Jackson Immunoresearch) for 2 h at room temperature in the dark. Immunofluorescence images were captured using a fluorescence microscope (Keyence BZ-X810, Osaka, Japan).