Paraformaldehyde-fixed, paraffin embedded (rat stomach); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (F-Actin) Polyclonal Antibody, Unconjugated (bs-1571R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat skeletal muscle); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (F-Actin) Polyclonal Antibody, Unconjugated (bs-1571R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Sample: Lane1:Hela (Human) Whole Cell Lysate at 30 ug
Lane2:Jurkat (Human) Whole Cell Lysate at 30 ug
Lane3:293T (Human) Whole Cell Lysate at 30 ug
Lane4:Hepg2 (Human) Whole Cell Lysate at 30 ug
Lane5:A431 (Human) Whole Cell Lysate at 30 ug
Lane6:A549 (Human) Whole Cell Lysate at 30 ug
Lane7:H9C2 (Rat) Whole Cell Lysate at 30 ug
Lane8:Testis (Mouse) Tissue Lysate at 30 ug
Primary: Anti-F-Actin(bs-1571R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD

Blank control (blue line): Hela (blue).
Primary Antibody (green line): Rabbit Anti- F-Actin antibody (bs-1571R)
Dilution: 3μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 80% methanol (5 min at -20℃) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature . Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.