Application Notes: A customer has reported negative results in live-cell applications for FACS, indicating that a fixation step may be required
Western blot analysis of mouse lung lysate using Claudin 5 antibody (dilution at 1:5000)
Blank control: K562. Primary Antibody (green line): Rabbit Anti-Claudin 5 antibody (orb182867), dilution: 2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 µg/Test. Protocol, The cells were 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 20000 events was performed.
Paraformaldehyde-fixed, paraffin embedded (Human kidney), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37C for 30 min, Antibody incubation with (Claudin 5) Polyclonal Antibody, Unconjugated (orb182867) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Protein: lung (mouse) lysates at 45 ug, Primary: Anti-Claudin 5 (orb182867) at 1:300, Secondary: HRP conjugated Goat-Anti-Rabbit IgG at 1:5000, ECL excitated the fluorescence, Predicted band size: 23 kD, Observed band size: 23kD.
Flow cytometric analysis of K562 cells using Claudin 5 antibody.
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