MCF-7 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37C for 20 min, Antibody incubation with (phospho-Rb (Ser780)) polyclonal Antibody, Unconjugated (orb11338) 1:100, 90 minutes at 37C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
MCF-7 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37C for 20 min, Antibody incubation with (phospho-Rb (Ser780)) polyclonal Antibody, Unconjugated (orb11338) 1:100, 90 minutes at 37C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
Molt-4 cells were fixed with 4% PFA for 10 min at room temperature, permeabilized with 90% ice-cold methanol for 20 min at -20C, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-Rb (Ser780) Antibody (orb11338) at 1:500 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Paraformaldehyde-fixed, paraffin embedded (mouse colon), 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 (phospho-Rb (Ser780)) Polyclonal Antibody, Unconjugated (orb11338) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: Lane 1: Mouse Thymus tissue lysates, Lane 2: Rat Thymus tissue lysates, Lane 3: Human Jurkat cell lysates, Primary: Anti-phospho-Rb (Ser780) (orb11338) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 106 kDa, Observed band size: 125 kDa.
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