Actingreen

Cells (5 × 10⁴ cells/mL) were plated onto polyacrylamide gel substrates and fixed in 4% paraformaldehyde for 15 min at room temperature. Then, the cells were washed with PBS, permeabilised with 0.1% Triton X-100 for 10 min, washed again with PBS, and incubated with ActinGreen (KeyGEN, BioTECH) for 40 min in the dark. The samples were washed with PBS and fluorescence images were obtained using an inverted optical microscope (Leica, Germany) with 488 laser lines. The obtained images were linearly analysed and pseudo-coloured using the ImageJ analysis software.

Cell samples were fixed with 4% paraformaldehyde. Liver tissue paraffin sections were dewaxed and placed into appropriate repair solutions for antigen repair according to antibody properties. Then the cell sample and the tissue sample were immunofluorescence stained in the same steps as below. The membrane was broken with 0.5% Triton-100 for about 15 minutes and then washed with PBS and closed with goat serum at room temperature for 30 minutes. The antibody was diluted in PBS solution according to the antibody instructions (anti-histone H3, CST, 4499, 1:400) and incubated overnight at 4°C. Then, the tissue sections were incubated with ActinGreen (KeyGEN BioTECH, KGMP001, 1:40) at room temperature for 20 minutes. Finally, the sections were incubated with secondary antibody (Cy3-AffiniPure goat anti-rabbit IgG, Wuhan Promoter Biological CO., LTD, 1:200) for 2 hours at room temperature and DAPI (Wuhan Promoter Biological Co., Ltd) for 15 minutes at room temperature before being photographed by fluorescence microscopy.

The cytoskeletal organization in ovarian cancer cells was investigated by confocal imaging. The cells were seeded into 35 mm cover glass bottom culture dishes (Nest) at a density of 5 × 10⁴ mL⁻¹ and cultured in the 37 °C incubator for 2 days prior to staining. Then, the culture medium was removed and 1 mL PBS was added to each culture dish. After washing three times with PBS, the samples with or without echinomycin treatments were fixed by 4% paraformaldehyde (PFA) for 15 min, and 0.1% Triton-X-100 was used for permeabilization. After that, the cells were stained with ActinGreen (KeyGEN BioTECH). A laser scanning confocal microscope (SP8, Leica) was used to image the cytoskeletal organization by F-actin. The fluorescence imaging was captured at 488 nm excitation wavelength. Moreover, the images were processed with the software Image J.