Sample:
Brain(Mouse) lysate at 30ug;
Liver(Mouse) lysate at 30ug;
Primary: Anti-HSP-27 (SL0730R) at 1:200;
Secondary: AP conjugated Goat Anti-Rabbit IgG(SL0295G-AP) at 1: 3000 dilutionNBT/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 (SL0730R) 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
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 (HSP27) Polyclonal Antibody, Unconjugated (SL0730R ) 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 stomach tissue); 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 (HSP27) Polyclonal Antibody, Unconjugated (SL0730R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat uterus); 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 (HSP27) Polyclonal Antibody, Unconjugated (SL0730R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human breast cancer); 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 (HSP27) Polyclonal Antibody, Unconjugated (SL0730R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.
Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-HSP-27 Polyclonal Antibody, Unconjugated(SL0730R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: rat brain tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-HSP-27 Polyclonal Antibody, Unconjugated(SL0730R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(SL0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,SLC0033) was used to stain the cell nuclei
Blank control (blue line): A431 cells (blue).
Primary Antibody (green line): Rabbit Anti-HSP27 antibody (SL0730R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 70% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. 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.
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