Alexa fluor 594 donkey anti mouse igg secondary antibody

Hypertrophic scar-derived fibroblasts in confocal culture dishes were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X100 (Sigma, USA) for 20 min and blocked with 5% donkey serum (Jackson, USA) for 1 h. After that, cells were incubated with mouse monoclonal anti-α-SMA primary antibody (Abcam, UK) at 4°C overnight. On the following day, cells were incubated with Alexa Fluor^® 594 donkey anti-mouse IgG secondary antibody (Invitrogen, USA) for 1 h at room temperature. The nuclei were stained with Hoechst (Invitrogen, USA). Zeiss laser-scanning microscope was used to analyze the fluorescence.

HepG2 cells (1.5 × 10⁵ cells/well) were allowed to adhere overnight to glass coverslips in a 6-well plate. After treatment, cells were fixed with 4% PFA for 30 min at room temperature. Fixed cells were incubated with 0.3% Triton X-100 in PBS, and blocked with 10% BSA in PBS for 1 h. The cells were incubated with the primary antibody against PFN1 (dilution 1:50; Cat. #: 3237; CST) or ACTN05 (C4) (1:200 dilution; Cat. #: ab3280; Abcam) overnight at 4°C followed by Alexa-Fluor 488-conjugated goat anti-rabbit IgG antibody (1:200 dilution; Cat. #: O-6381; Invitrogen) or Alexa Fluor 594 donkey anti-mouse IgG secondary antibody (1:200 dilution; Cat. #: A-21203; Invitrogen) for 1 h at room temperature. Alexa Fluor 555^®-phalloidin (1:20 dilution; Cat. #: 8953; CST) and 4′6-diamidino-2-phenylindole (DAPI, Cat. #: P36931; Invitrogen) staining were then used to stain F-actin and nucleus, respectively. Immunofluorescence images were visualized using an inverted fluorescent microscope (Olympus, Japan).