Fluormount with dapi

MH-S cell were placed on slides, fixed with 4% paraformaldehyde at room temperature for 30 min, then washed with PBS for 2–3 times. Placed the cell slides in 1% Triton x-100 (JISSKANG, China) for 10 min. After washing with PBS, the cell slides were blocked in 5% BSA for 1 h. Then the diluted anti-mouse caspase-11(ab240991, abcam, 1:100) was added and incubated overnight at 4 °C. On the second day, incubate the cell slides with the fluorescent secondary antibody (Beyotime, China) at 37 °C for 1 h under the dark environment. After rinsing, fluormount with DAPI (AntGene, China) was added to stain the nucleus. Finally, seal the tablet and observe the slides under the fluorescence microscope.

Cells were washed three times with PBS, fixed with precooled 4% paraformaldehyde for 15 min at room temperature, permeabilized for 10 min with 0.1% Triton X-100, and blocked for 30 min with 5% bovine serum albumin (BSA) in PBS. Next, the cells were incubated with primary antibody against the specific protein at 4 °C overnight, which was followed by incubation with Alexa Fluor-labeled secondary antibody (Molecular Probes) for 2 h at room temperature. Cell nuclei were subsequently stained with Fluormount with DAPI (antGene). The fluorescence was observed by a confocal laser-scanning microscope (Leica TCS SP2). EGFP and Alexa 488 were excited at a wavelength of 488 nm, Cy3 was excited at 561 nm, and DAPI was excited at 408 nm.