Sample:
Lane 1: MCF-7 (Human) Cell Lysate at 30 ug
Lane 2:U251 (Human) Cell Lysate at 30 ug
Primary: Anti-Phospho-HSP27 (Ser15) (SL3225R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27 kD
Observed band size: 27 kD
Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma); 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 (P-HSP27 (Ser15)) Polyclonal Antibody, Unconjugated (SL3225R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (Phospho-HSP27 (Ser15)) polyclonal Antibody, Unconjugated (SL3225R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control (blue line): A431 cells (fixed with 70% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃).
Primary Antibody (green line): Rabbit Anti-Phospho-HSP27 (Ser15) antibody (SL3225R),Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC,Dilution: 1μg /test.
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