Sample:Optic nerve (Rat)cell Lysate at 40 ug
Primary: Anti-GFAP(bs-0199R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-RabbitIgG at 1/20000 dilution
Predicted band size: 48 kD
Observed band size: 53 kD

Sample: U251 Cell Lysate at 40 ug
Primary: Anti- GFAP (bs-0199R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/10000 dilution
Predicted band size: 48 kD
Observed band size: 50 kD

Sample:
Cerebellum (Rat) Lysate at 40 ug
Cerebellum (Mouse) Lysate at 40 ug
Eye (Mouse) Lysate at 40 ug
Primary: Anti-GFAP (bs-0199R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 48 kD
Observed band size: 48 kD

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-GFAP Polyclonal Antibody, Unconjugated(bs-0199R) 1:400, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-GFAP Polyclonal Antibody, Unconjugated(bs-0199R) 1:500, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) 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 cerebellum); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:300 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (YF488) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (human brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:500 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (YF488) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:500 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (YF488) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:500 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (YF488) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:500 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (YF488) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-FITC) for 90 minutes, and DAPI for nuclei staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6) 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 (GFAP) Polyclonal Antibody, Unconjugated (bs-0199R) at 1:200 overnight at 4°C, followed by a conjugated secondary (bs-0295G-Cy3) at [1:500] for 90 minutes and DAPI staining of the nuclei.

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,C-0005) at 37℃ for 20 min;
Incubation: Anti-GFAP Polyclonal Antibody, Unconjugated(bs-0199R) 1:400, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Blank control:U87MG.
Primary Antibody (green line): Rabbit Anti-GFAP antibody (bs-0199R)
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).
Primary Antibody:Rabbit Anti- GFAP antibody(bs-0199R), 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-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice. Primary antibody (bs-0199R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.