Alexa fluor cy5

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).

Chromosome spreads were performed according to the procedure described previously (Li et al., 2019 (link)). Briefly, after proper removal of zona pellucida, the oocytes were washed in prewarmed M2 medium for a brief recovery; subsequently, the oocytes were exposed to spread solution (1% paraformaldehyde in distilled H₂O (pH 9.2) containing 0.15% Triton X-100 and 3 mM dithiothreitol) on a clean glass slide, as previously reported (Hodges and Hunt, 2002 (link)). After drying slowly in a half-open humidified chamber at room temperature (RT), the fixed oocytes were blocked with 2% BSA in PBS for 1 h at RT. For immunofluorescent staining, the oocytes were then incubated with primary antibodies overnight at 4°C. After three washes (10 min each wash), the slides were then incubated with corresponding secondary antibody for 2 h at RT. Primary human anti-ACA (anticentromere antibody) antibody (1:50; 15–234, Antibodies Incorporated) was used for detecting centromeres with a corresponding secondary antibody conjugated with Alexa Fluor Cy5 (709-175-149, Jackson ImmunoResearch).