Anti-ZAP70 antibody [EPR29942-36]

• Flow Cyt

Lab

Flow Cytometry - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

Flow cytometry overlay histogram showing top CD3⁺ human PBMCs (positive cells) and bottom CD19⁺ human PBMCs (negative cells) stained with ab325911 (red line). Cells were fixed and permeabilised with BD Cytofix/Cytoperm for 20 min. Cells were incubated for 30 min on ice in 1× PBS containing 10 μg/ml human IgG and 10% normal goat serum to block Fc receptors and non‑specific protein-protein interactions, followed by the antibody ab325911 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1×10⁶ cells in 100 μl at 0.2 μg/ml) for 30 min at 22°C. The cells were simultaneously stained with CD3 and CD19.

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control, used at the same concentration and under the same conditions as the primary antibody. An unlabelled sample (without incubation with the primary antibody, blue line) was also used as a control. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor 488), preadsorbed, was incubated at 1/4000 for 30 min at 22°C.

Acquisition of >30, 000 events was performed using a 50 mW blue laser (488 nm) and a 525/40 bandpass filter. Events were gated on either CD3⁺ or CD19⁺ cells.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

ab325911 staining ZAP70 in Jurkat WT/KO cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal rabbit serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab325911 at 1 μg/mL (shown in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor 488), pre-adsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

A subpopulation of ZAP70 positive cells display nuclear subcellular staining, as shown in PMID : 9362531 and PMID : 15133473.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

ab325911 staining ZAP70 in Human PBMCs cells. The cells were fixed with BD Cytofix/Cytoperm™ for 20 min, permeabilized with for 5 minutes and then incubated in 1× PBS containing 10 μg/ml human IgG and 10% normal goat serum to block Fc receptors and non‑specific protein-protein interactions in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab325911 at 1μg/mL (shown in green), anti-human CD3 (APC) (shown in pseudocolour magenta) and anti-human CD19 (PE) (shown in pseudocolour blue). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor 488), pre-adsorbed at 1/1000 dilution. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

ZAP70 is expressed on CD3 positive PBMCs, whilst ZAP70 and CD19 expression is mutually exclusive. A subpopulation of ZAP70 positive PBMCs display nuclear subcellular staining, as shown in PMID : 9362531 and PMID : 15133473.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

ab325911 staining ZAP70 in Human PBMCs cells. The cells were fixed with BD Cytofix/Cytoperm™ for 20 min, permeabilized with for 5 minutes and then incubated in 1× PBS containing 10 μg/ml human IgG and 10% normal goat serum to block Fc receptors and non‑specific protein-protein interactions for 1h. The cells were then incubated overnight at 4°C with ab325911 at 1 μg/mL (shown in green), anti-human CD19 (APC) (shown in pseudocolour magenta) and anti-human CD56 (PE) (shown in pseudocolour blue). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor 488), pre-adsorbed at 1/1000 dilution. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

ZAP70 is expressed on CD56 positive PBMCs, whilst ZAP70 and CD19 expression is mutually exclusive. A subpopulation of ZAP70 positive PBMCs display nuclear subcellular staining, as shown in PMID : 9362531 and PMID : 15133473.

• Flow Cyt

Lab

Flow Cytometry - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

Flow cytometry overlay histogram showing top CD3⁺ C57BL/6 mouse splenocytes (positive cells) and bottom CD11b⁺ C57BL/6 mouse splenocytes (negative cells) stained with ab325911 (red line). Cells were fixed and permeabilised with BD Cytofix/Cytoperm for 20 min. Cells were incubated for 30 min on ice in 1× PBS containing 10 μg/ml anti‑CD16/CD32 and 10% normal goat serum to block Fc receptors and non‑specific protein-protein interactions, followed by the antibody ab325911 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1×10⁶ cells in 100 μl at 1μg/ml) for 30 min at 22°C. The cells were simultaneously stained with CD3 and CD11b.

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control, used at the same concentration and under the same conditions as the primary antibody. An unlabelled sample (without incubation with the primary antibody, blue line) was also used as a control. The secondary antibody Goat Anti‑Rabbit IgG H&L (Alexa Fluor 488), preadsorbed, was incubated at 1/4000 for 30 min at 22°C.

Acquisition of >30, 000 events was performed using a 50 mW blue laser (488 nm) and a 525/40 bandpass filter. Events were gated on either CD3⁺ or CD11b⁺ cells.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [EPR29942-36] (AB325911)

ab325911 staining ZAP70 in mouse splenocytes cells. The cells were fixed and permeabilized with BD Cytofix/Cytoperm™ for 20 min and then incubated in 1× PBS containing 10 μg/ml anti‑CD16/CD32 and 10% normal goat serum to block Fc receptors and non‑specific protein-protein interactions for 1 hr. The cells were then incubated overnight at 4°C with ab325911 at 1 μg/mL (shown in pseudocolour magenta), anti-mouse CD3 (PE) at 1/2000 dilution (shown in pseudocolour blue) and anti-mouse/human CD11b (Alexa Fluor 488) (shown in green) at 1/500 dilution. Cells were then incubated with ab150083 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) at 1/1000 dilution.
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

ZAP70 is expressed on CD3 positive splenocytes, whilst ZAP70 and CD11b expression is mutually exclusive. A subpopulation of ZAP70 positive splenocytes display nuclear subcellular staining, as shown in PMID : 9362531 and PMID : 15133473.