Alexa fluor 488 conjugated anti mouse igg h l secondary antibody

HUVECs were fixed with 4% paraformaldehyde for 30 min, washed with PBS for 5 min, and permeabilized in 0.1% Triton X-100 at room temperature for 10 min. The cells were incubated with rabbit polyclonal antibody against HO-1 (1:100), mouse monoclonal antibody against NPM1 (1:100), rabbit polyclonal antibody against fibrillarin (1:100) or mouse monoclonal antibody against p53 (1:50, Cell Signaling Technology, #2524) at 4 °C overnight. The cells were then washed with PBS and incubated with appropriate secondary antibodies in goat serum (Boster Biological Technology, California, USA) for 1 h at room temperature. Alexa Fluor 594-conjugated anti-rabbit IgG (H+L) secondary antibody and Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (Proteintech, SA00013-4, SA00013-1) were used. Nuclei were stained with 5 μg/ml DAPI (Sigma). Images were acquired using cell auto imaging system (EVOS FL Auto, Life Technologies) or FV3000 laser scanning confocal microscope (Olympus Life Science).

HUVECs were cultured on 48-well chamber slides and stimulated with H₂O₂ (50 μM) for 1 h and grown for 48 h to induce senescent cells. Cells were then treated with 10 μM Hemin or the vehicle (Saline) for 48 h. After 2 days, cells were fixed with 4% (w/v) paraformaldehyde for 30 min, washed with PBS for 5 min, and permeabilized in 0.1% (w/v) Triton X-100 at room temperature for 10 min. The cells were incubated with mouse monoclonal antibody against eNOS (1:50), rabbit polyclonal antibody against HO-1 (1:100) or rabbit polyclonal antibody against Akt (1:50) at 4 °C overnight. After incubation with Alexa Fluor 594-conjugated anti-rabbit IgG (H+L) secondary antibody and Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (Proteintech, Manchester, UK) for 1 h at room temperature, nuclei were stained with DAPI (5 mg/ml, Sigma-Aldrich, St. Louis, MO) and analyzed by cell auto imaging system (EVOS FL Auto, Life Technologies, New York, USA).