4 6 diamidino 2 phenylindole dapi blue

AGS cells or 4-μm sections of xenograft tumor tissue were plated on a slide glass, which was dried for 15 min at room temperature and fixed with 3.7% formaldehyde. After washing with water, slides were incubated with a rabbit monoclonal anti-human SHP-1 IgG antibody (Abcam, ab124942, Cambridge, UK), a rabbit monoclonal anti-human vimentin IgG antibody, a rabbit polyclonal anti-human E-cadherin IgG antibody, a mouse monoclonal anti-human Snail1 IgG antibody or a mouse monoclonal anti-human p-STAT3 IgG antibody (1/100 dilution) at 4 °C overnight. After washing, slides were incubated with goat anti-mouse IgG (green) or goat anti-rabbit IgG (green) (1/500 dilution) at room temperature for 1 h. Counterstaining was performed by incubating with 4′,6-diamidino-2-phenylindole (DAPI, blue) (Vectashield, Vector Laboratories, Burlingame, CA, USA) at room temperature for 5 min. Slides were observed under a confocal microscope (LSM 700, Carl Zeiss, Oberkochen, Germany) and images were captured by using a high-resolution digital camera (Carl Zeiss).

BV2 cells were mock-treated, infected with ZIKV_PE243 or ZIKV_MR766, with an MOI of 1, or stimulated with LPS + ATP as a positive control. After 24, 48, and 72 hpi, cells were fixed and permeabilized as described previously. Then, the cells were incubated overnight with the primary anti-Iba1 antibody at 1:500 (Wako, code 019-19741), anti-CD68 antibody at 1:500 (Bio-Rad, MC1957, Hercules, CA, USA), or with anti-MHC II conjugated with phycoerythrin—PE (eBioscience, San Diego, CA, USA, ref. 12-5322-81). The cells were then stained with the respective anti-IgG secondary antibodies conjugated to AlexaFluor488 or AlexaFluor647. To stain cell nuclei, a mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (blue) (Vectashield—Vector Laboratories, Mowry Ave Newark, CA, USA) was used. Fluorescence microscopy was performed simultaneously with the same antibody solutions, and all micrographs were taken with the same settings using a Zeiss LSM 710 confocal microscope equipped with a 63× objective and digital imaging system (Roper Scientific camera and Zen (black edition) software).

AGS cells were treated with 10 μM ATO for 24 h and plated on a glass slide, air-dried for 15 min at room temperature and fixed with 3.7% formaldehyde. After washing, slides were incubated with a mouse monoclonal antihuman Snail1 IgG antibody or a rabbit polyclonal antihuman E-cadherin IgG antibody with 1/100 dilution during overnight. And then, slides were washed and incubated with goat antimouse IgG (red, #A-11004) or goat antirabbit IgG (green, #A-11008) for 1 h with 1/100 dilution. Secondary antibodies were purchased from Invitrogen (Waltham, MA, USA). Couterstaining was performed by using 4′,6-diamidino-2-phenylindole (DAPI, blue) (Vectashield, Vector Laboratories, Burlingame, CA, USA) for 5 min. Cells were observed by using a confocal microscope (LSM 700, Carl Zeiss, Oberkochen, Germany) and pictures were captured with digital camera (Carl Zeiss).