Sample:
Uterus(Mouse)Lysate at 40 ug
Ovary (Mouse)Lysate at 40 ug
Primary: Anti-GLI1(bs-1206R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-RabbitIgG at 1/20000 dilution
Predicted band size: 118kD
Observed band size: 130kD

Sample:HepG2 (Human)Cell Lysate at 40 ug
Primary: Anti-GLI1(bs-1206R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-RabbitIgG at 1/20000 dilution
Predicted band size: 118kD
Observed band size: 130kD

Paraformaldehyde-fixed, paraffin embedded (Rat testis); 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 (GLI1) Polyclonal Antibody, Unconjugated (bs-1206R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse brain); 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 (GLI1) Polyclonal Antibody, Unconjugated (bs-1206R) 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 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 (GLI1) Polyclonal Antibody, Unconjugated (bs-1206R) 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 brain); 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 (GLI1) Polyclonal Antibody, Unconjugated (bs-1206R) 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 brain glioma); 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 (GLI1) Polyclonal Antibody, Unconjugated (bs-1206R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control:Hela.
Primary Antibody (green line): Rabbit Anti-GLI1 antibody (bs-1206R)
Dilution: 1ug/Test;
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 0.5ug/Test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃.The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 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).