Sample:
Lane 1: Mouse Stomach tissue lysates
Lane 2: Mouse Spleen tissue lysates
Primary: Anti-Caspase-9 (bs-0049R) 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

Sample:
293T-UV Cell (Human) Lysate at 30 ug
Primary: Anti-Caspase-9 (Bs- 0049R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35/50 kD
Observed band size: 35/50 kD

Paraformaldehyde-fixed, paraffin embedded (rat pancreas); 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 (Caspase-9) Polyclonal Antibody, Unconjugated (bs-0049R) 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 lung); 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 (Insulin like growth factor 1 ) Polyclonal Antibody, Unconjugated (bs-0014R) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody (sp-0023) for 20 minutes and DAB staining.

Tissue/cell: human colon carcinoma; 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-Caspase-9 Polyclonal Antibody, Unconjugated(bs-0049R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-Caspase-9 Polyclonal Antibody, Unconjugated(bs-0049R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: MCF-7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Caspase-9) Polyclonal Antibody, Unconjugated (bs-0049R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-Cy3) at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Tissue/cell: HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (bs-0049R) 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, C02-04002) was used to stain the cell nuclei.

Tissue/cell: Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (bs-0049R) 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, C02-04002) was used to stain the cell nuclei.

HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Caspase-9) polyclonal Antibody, Unconjugated (bs-0049R) 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: K562 (blue).
Primary Antibody:Rabbit Anti-caspase-9 antibody (bs-0049R,Green); Dilution: 1μg 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 (bs-0049R, 1μg /1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (30min) 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. Acquisition of 20,000 events was performed.

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: 1μg in 100 μL1X PBS containing 0.5% BSA(green).