Anti-CD80 antibody [EPR22183]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD80 antibody [EPR22183] (AB225674)

Immunofluorescent analysis of 100% methanol-fixed Raji (human Burkitt's lymphoma cell line) and HUVEC (human umbilical vein endothelial cell line) cells labeling CD80 with ab225674 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Raji cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

PBS only control : Used PBS instead of primary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution dilution.

Negative control : HUVEC (PMID : 15064090).

• Flow Cyt

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Flow Cytometry - Anti-CD80 antibody [EPR22183] (AB225674)

Flow cytometric analysis of HUVEC (human umbilical vein endothelial cell line) (left panel) cells and Raji (human Burkitt's lymphoma cell line)(right panel) cells labeling CD80 with ab225674 at 1/400 (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Negative control : HUVEC (PMID : 15064090).

• Flow Cyt

Lab

Flow Cytometry - Anti-CD80 antibody [EPR22183] (AB225674)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with CD3/CD28 activation (bottom), with ab225674 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left).

PBMCs were incubated for 30 min on ice in 1x 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 ab225674 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 5.0 μg/ml (1/400)) for 30 min at 22°C . Cells were simultaneously stained with CD4

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/2000 for 30min at 22°C.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IP

Unknown

Immunoprecipitation - Anti-CD80 antibody [EPR22183] (AB225674)

CD80 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab225674 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab225674 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : Raji whole cell lysate 10 μg (Input).
Lane 2 : ab225674 IP in Raji whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab225674 in Raji whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 1 second.

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Immunoprecipitation - Anti-CD80 antibody [EPR22183] (ab225674)

Predicted band size: 33 kDa

Observed band size: 60 kDa

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