Dylight 488 conjugated goat anti rabbit igg antibody

HT-29 monolayers were cultured in 24-well plates and treated as described in the western blotting assay. Monolayers were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 10 min. Then cells were blocked with 1% bovine serum albumin (BSA) in PBST for 30 min followed by incubation with rabbit anti-MUC2 antibody overnight at 4°C. Cells were washed four times with PBST and incubated with DyLight 488-conjugated goat anti-rabbit IgG antibody (Abbkine, United States) at room temperature for 1 h followed by incubation with Hoechst 33342 for 10 min. Results were observed using fluorescence microscopy (Nikon Eclipse: TE 2000-E, Japan). Each assay was performed in triplicate wells and repeated three times.

STEC grown on coverslips were washed three times with PBS after different treatments. The cells were fixed in cold methanol for 20 min and blocked with 3% BSA for 2 h. Cells were incubated with anti-vimentin rabbit polyclonal antibody (1:400, Proteintech, Wuhan, China) at 4 °C overnight and then with DyLight 488-conjugated goat anti-rabbit IgG antibody (1:400, Abbkine, Wuhan, China) at RT for 1 h. The nuclei were stained with 4',6-Diamidino-2-phenylindole (DAPI, Beyotime, Nanjing, China). The coverslips were mounted using ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) and imaged with a fluorescence microscope (Axio Observer 7; LD Plan-NEOFLUAR 40 × /0.6; ZEISS, Germany).

Cultured NP cells were rinsed three times using phosphate-buffered saline (PBS), fixed by 4% formaldehyde for 15 min, incubated in 0.25% Triton X‐100 for 15 min, and blocked by 5% bovine serum albumin (BSA, Sigma, Ohio, USA) in PBS for 30 min at room temperature. Then, the cells were treated with primary antibody against p16 (ab51243, Abcam, Cambridge, UK) for one night at 4°C, and incubated with DyLight 488-conjugated goat anti-rabbit IgG antibody (Abbkine, California, USA) for 2 hr in the dark at room temperature. The cells were visualized using a fluorescence microscope (Leica, Wetzlar, Germany), and nuclei were counterstained with DAPI (Beyotime, Shanghai, China).