Antibody Type: Rabbit Polyclonal

Application: WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

Reactivity: Human,Mouse,Rat,Dog (predicted:Cow)

IgG

Human,Mouse,Rat,Dog (predicted:Cow)

1. Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-Caspase-9 Polyclonal Antibody, Unconjugated (TMAB-00292) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
2. Tissue/cell: human colon carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-Caspase-9 Polyclonal Antibody, Unconjugated (TMAB-00292) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
3. Blank control: RSC96 (blue), the cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice.
Isotype Control Antibody: Rabbit Igg (orange);
Secondary Antibody: Goat anti-rabbit IgG-Pe (white blue),
Dilution: 1:200 in 1 X PBS containing 0.5% BSA;
Primary Antibody Dilution: 1 μg in 100 μL 1X PBS containing 0.5% BSA (green).
4. Blank control: K562 (blue). Primary Antibody: Rabbit Anti-caspase-9 antibody (TMAB-00292,Green); Dilution: 1 μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit Igg (orange),used under the same conditions; Secondary Antibody: Goat anti-rabbit IgG-FITC (white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 80% methanol (5 min) and and then permeabilized with 0.01M PBS-Tween for 20 min. Primary antibody (TMAB-00292, 1 μg/1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (30 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/FITC antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min at room temperature.
5. Paraformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Insulin like growth factor 1) Polyclonal Antibody, Unconjugated at 1:400 overnight at 4°C, followed by a conjugated secondary antibody for 20 min and DAB staining.
6. Tissue/cell: Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (TMAB-00292) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
7. Paraformaldehyde-fixed, paraffin embedded (rat pancreas); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Caspase-9) Polyclonal Antibody, Unconjugated (TMAB-00292) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
8. Tissue/cell: HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (TMAB-00292) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
9. Tissue/cell: MCF-7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Caspase-9) Polyclonal Antibody, Unconjugated (TMAB-00292) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
10. HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (TMAB-00292) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
11. Sample:
293T-UV Cell (Human) Lysate at 30 μg
Primary: Anti-Caspase-9 at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35/50 kDa
Observed band size: 35/50 kDa
12. Sample:
Lane 1: Mouse Stomach tissue lysates
Lane 2: Mouse Spleen tissue lysates
Primary: Anti-Caspase-9 (TMAB-00292) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35/50 kDa
Observed band size: 52 kDa

WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100; IF: 1:100-500; FCM: 1μg/test

Polyclonal

KLH conjugated synthetic peptide: human Caspase-9 subunit p35

Human

842

Caspases,Cytochrome C,Metabolism,Caspases,Cytochrome C,Caspases,Apoptosis