Antibody Type: Rabbit Polyclonal

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

Reactivity: Human,Rat

IgG

Human,Rat

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-osteopontin Polyclonal Antibody, Unconjugated (TMAB-01298) 1:300, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
2. Tissue/cell: rat shin bone; 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-osteopontin Polyclonal Antibody, Unconjugated (TMAB-01298) 1:300, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
3. Sample:
Lane 1: HepG2 (Human) Cell Lysate at 30 μg
Lane 2: A549 (Human) Cell Lysate at 30 μg
Lane 3: U251 (Human) Cell Lysate at 30 μg
Primary: Anti-Osteopontin (TMAB-01298) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 34 kDa
Observed band size: 60 kDa
4. 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 (Osteopontin) Polyclonal Antibody, Unconjugated (TMAB-01298) at 1:400 overnight at 4°C, followed by a conjugated secondary for 20 min and DAB staining.
5. Blank control: HepG2. Primary Antibody (green line): Rabbit Anti-Osteopontin antibody (TMAB-01298)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF647
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at room temperature. 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.

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

WB: 1:500-2000; IHC-P: 1:500-2000; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 1ug/Test

Polyclonal

KLH conjugated synthetic peptide: mouse OPN

Mouse

20750