Anti-CD40 antibody [EPR20735]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD40 antibody [EPR20735] (AB224639)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab224639 (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 interaction followed by the antibody ab224639 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 μl at 0.2 μg/ml (1/10550)) for 30min on ice. The cells were simultaneously stained with CD19.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (AB224639)

Tissue Microarrays stained for "Anti-CD40 antibody [EPR20735]" using "ab224639"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab224639 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (AB224639)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling CD40 with ab224639 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and weakly cytoplasmic staining in Raji cell line.

Negative control : Jurkat T cell line (PMID : 10498643).

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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

• Flow Cyt

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Flow Cytometry - Anti-CD40 antibody [EPR20735] (AB224639)

Flow cytometric analysis of Raji (human Burkitt's lymphoma cell line) cell line labeling CD40 with ab224639 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled 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 total viable cells.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (AB224639)

ab224639 staining CD40 in wild-type U2OS cells (top panel) and CD40 knockout U2OS cells (bottom panel). The cells were fixed with PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab224639 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (AB224639)

Immunohistochemical analysis of paraffin-embedded human Hodgkin's lymphoma tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in Reed-Sternberg cells and tumor cells of human Hodgkin's lymphoma (PMID : 7507299) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (AB224639)

Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining in human diffuse large B-cell lymphoma tissue (PMID : 20593977) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (AB224639)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in human tonsil tissue (PMID : 20593977; PMID : 7507299) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

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Immunoprecipitation - Anti-CD40 antibody [EPR20735] (AB224639)

CD40 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab224639 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224639 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1 : Raji whole cell lysate 10 μg (Input).
Lane 2 : ab224639 IP in Raji whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab224639 in Raji whole cell lysate (-).

Exposure time : 3 minutes.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD40 antibody [EPR20735] (ab224639)

Predicted band size: 30 kDa

Observed band size: 42 kDa

true

• WB

Supplier Data

Western blot - Anti-CD40 antibody [EPR20735] (AB224639)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD40 antibody [EPR20735] (ab224639) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

Human lymphoma tissue lysate at 20 µg

Lane 3:

Raji (human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 30 kDa

Observed band size: 42 kDa

true

Exposure time: 15s