HSP27 Rabbit Polyclonal Antibody

Blank control (blue line): A431 cells (blue). Primary Antibody (green line): Rabbit Anti-HSP27 antibody (orb10845), Dilution: 2 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC, Dilution: 1 µg/Test. Protocol, The cells were fixed with 70% methanol (Overnight at 4°C) and then permeabilized with 90% ice-cold methanol for 20 min at -20°C. 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 20000 events was performed.

Paraformaldehyde-fixed, paraffin embedded (Human breast cancer), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (HSP27) Polyclonal Antibody, Unconjugated (orb10845) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat skeletal muscle), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (HSP27) Polyclonal Antibody, Unconjugated (orb10845) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat stomach tissue), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (HSP27) Polyclonal Antibody, Unconjugated (orb10845) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Rat uterus), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (HSP27) Polyclonal Antibody, Unconjugated (orb10845) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Brain (Mouse) lysate at 30 ug, Liver (Mouse) lysate at 30 ug, Primary: Anti-HSP-27 (orb10845) at 1:200, Secondary: AP conjugated Goat Anti-Rabbit IgG at 1:3000 dilution, NBT/BCIP staining, Predicted band size: 27kD, Observed band size: 27kD.

Sample: Lane 1: Hela (Human) Cell Lysate at 30 ug, Lane 2: MCF-7 (Human) Cell Lysate at 30 ug, Lane 3: A431 (Human) Cell Lysate at 30 ug, Lane 4: Huvec (Human) Cell Lysate at 30 ug, Lane 5: HepG2 (Human) Cell Lysate at 30 ug, Primary: Anti-HSP27 (orb10845) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 27-30 kD, Observed band size: 27 kD.

Tissue/Cell: rat brain tissue, 4% Paraformaldehyde-fixed and paraffin-embedded, Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min, Blocking buffer (normal goat serum) at 37°C for 20 min, Incubation: Anti-HSP-27 Polyclonal Antibody, Unconjugated (orb10845) 1:200, overnight at 4°C, The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated (orb868589) used at 1:200 dilution for 40 minutes at 37°C. DAPI (5 ug/ml, blue) was used to stain the cell nuclei.