Blank control (blue line): A431 cells (fixed with 70% methanol (Overnight at 4C) and then permeabilized with 90% ice-cold methanol for 20 min at -20C). Primary Antibody (green line): Rabbit Anti-Phospho-HSP27 (Ser78) antibody (orb5481), dilution: 0.2 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.
Hela cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37C for 20 min, Antibody incubation with (Phospho-HSP27 (Ser78)) polyclonal Antibody, Unconjugated (orb5481) 1:100, 90 minutes at 37C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded (Rat heart), 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 37C for 30 min, Antibody incubation with (Phospho-HSP27 (Ser78)) Polyclonal Antibody, Unconjugated (orb5481) at 1:400 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: Lane 1: MCF-7 (Human) Cell Lysate at 30 ug, Lane 2: U251 (Human) Cell Lysate at 30 ug, Lane 3: U2os (Human) Cell Lysate at 30 ug, Lane 4: A431 (Human) Cell Lysate at 30 ug, Lane 5: Huvec (Human) Cell Lysate at 30 ug, Lane 6: A549 (Human) Cell Lysate at 30 ug, Primary: Anti-Phospho-HSP27 (Ser78) (orb5481) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 27 kD, Observed band size: 27 kD.
Sample: Muscle (Mouse) Lysate at 40 ug, Primary: Anti-Phospho-HSP27 (Ser78) (orb5481) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 23 kD, Observed band size: 23 kD.
IHC-P analysis of Rat heart using Phospho-HSP27 Ser78 Polyclonal Antibody at 1:400 dilution.
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