Cy3 labeled goat anti rabbit secondary antibody

The distribution of E-cadherin and NF-κB p65 in SW480 cells was determined by immunofluorescence staining. Cells grown on the coverslips were fixed and permeabilized in 0.1% Triton X-100. After blocking with normal goat serum (Solarbio), the cell slides were incubated in anti-E-cadherin or anti-p65 antibody (dilution 1:200; Boster) at 4°C overnight. Subsequently, the cells were incubated with Cy3-labeled goat anti-rabbit secondary antibody (1:200; Beyotime Institute of Biotechnology) for 1 hour. After washing off the unbound secondary antibodies, all slides were counterstained with 4′,6-diamidino-2-phenylindole. The images of stained slides were visualized under a laser scanning confocal microscope (Olympus, Tokyo, Japan)

In the present study, the primary trophoblast slides were first fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Goat serum (purchased from Solarbio, Beijing, China) was added in order to block non-specific binding sites, followed by incubation with CK18 antibody (dilution 1:50; #sc-6259; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), vimentin antibody (dilution 1:100; #WL0274; Wanleibio, Shenyang, China) and hPL antibody (dilution 1:100; #ab11396; Abcam, Cambridge, MA, USA) at 4°C overnight. The slides were then washed three times with PBS for 5 min and incubated with Cy3-labeled goat anti-rabbit secondary antibody (#A0516) or Cy3-labeled goat anti-mouse secondary antibody (#A0521) (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at room temperature. Subsequently, 4′,6-diamidino-2-phenylindole (DAPI) was added in order to stain nuclei, and they were then examined by fluorescence microscopy (BX53; purchased from Olympus, Tokyo, Japan).

The sections were blocked with goat serum for 15 min and then incubated with the primary antibody (p65, 1:200, Affinity) at 4°C overnight. After washed three times with PBS (5 min/time), the sections were incubated with Cy3-labeled goat anti-rabbit secondary antibody (1:200, Beyotime, Shanghai, China) at room temperature for 1 h. After washed three times with PBS (5 min/time), the nucleus was counterstained by DAPI (Aladdin, Shanghai, China). Finally, the stained sections were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan, magnification, ×400).

Paraffin sections of mouse colon tissues with a thickness of 5 μm were generated. The sections were incubated with 1:100 diluted CD4+ antibody (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C, and then with 1:200 diluted Cy3-labeled goat anti-rabbit secondary antibody (Beyotime). After secondary incubation at room temperature for 1 h, the nuclei were counter-stained with 4',6-diamidino-2-phenylindole. The sections were mounted with anti-fading reagent, observed under a fluorescence microscope (BX53, Olympus, Tokyo, Japan), and imaged under 400× magnification lens.

The cells were cultured on glass coverslips and fixed in 4% paraformaldehyde for 15 min. Slips were washed in PBS 3 times and incubated with 0.1% Triton X-100 for 30 min at room temperature. After a washing stage, the slips were blocked in goat serum for 15 min at room temperature. The cells were then incubated with anti-β-catenin antibody (1:200, BA0426; Boster, Wuhan, China) at 4°C overnight. Subsequently, the cells were washed with PBS and incubated with cy3-labeled goat anti-rabbit secondary antibody (1:200, Beyotime Institute of Biotechnology) at room temperature for 1 h. The cells were then co-stained with DAPI and then observed under a fluorescence microscope (BX53; Olympus).