Anti-CD40 antibody [RM1193]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [RM1193] (AB317586)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Raji (human Burkitt's lymphoma B lymphocyte) cells labelling CD40 with ab317586 at 1/50 (10.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing membranous staining in Raji cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : Jurkat (PMID : 10498643, PMID : 15708600).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Anti-CD40 antibody [RM1193] (AB317586)

Flow cytometric analysis of Jurkat(human T cell leukemia T lymphocyte from peripheral blood, Left) / Raji(human Burkitt's lymphoma B lymphocyte, Right) cells labelling CD40 with ab317586 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cell.
Negative control : Jurkat.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Human diffuse large B-cell lymphoma tissue labeling CD40 with ab317586 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human diffuse large B-cell lymphoma. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Human hodgkin's lymphoma tissue labeling CD40 with ab317586 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human hodgkin's lymphoma. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CD40 with ab317586 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human colon in (A) ab317586, non-specific staining on human colon in (B) ab13545. The image on (A) ab317586 is applied with Anti-CD40 antibody at 1/2000 dilution and (B) ab13545 is applied with Anti-CD40 antibody at 1/4000 dilution.
The section was incubated with ab317586 or ab13545 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD40 with ab317586 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil in (A) ab317586 and (B) ab13545.
The image on (A) ab317586 is applied with Anti-CD40 antibody at 1/2000 dilution and (B) ab13545 is applied with Anti-CD40 antibody at 1/4000 dilution.
The section was incubated with ab317586 or ab13545 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling CD40 with ab317586 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human cardiac muscle. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-CD40 antibody [RM1193] (AB317586)

CD40 was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab317586 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317586 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lane 2 : ab317586 IP in Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317586 in Raji whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CD40 antibody [RM1193] (ab317586) at 1/30 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate

Lane 2:

ab317586 IP in Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 111s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling CD40 with ab317586 at 1/500 (1.016 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling CD40 with ab317586 at 1/500 (1.016 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse cardiac muscle. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [RM1193] (AB317586)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD40 with ab317586 at 1/500 (1.016 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The section was incubated with ab317586 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

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Western blot - Anti-CD40 antibody [RM1193] (AB317586)

Anti-CD40 antibody (ab317586) staining at 1/1000 dilution is specific to human and mouse species. Anti-CD40 antibody (ab13545) at 1/1000 dilution is bandy than ab317586.

The molecular weight observed is consistent with the literature (PMID : 23404288).

Negative control : Jurkat (PMID : 10498643; PMID : 15708600).

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

Exposure time : Lane 1, 6 : 103 seconds Lane 2-5 : 92 seconds

All lanes:

Western blot - Anti-CD40 antibody [RM1193] (ab317586) at 1/1000 dilution

Lane 1:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 2:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 5:

RAW 264.7 treated with /ml LPS (Lipopolysaccharide) for 6 hours whole cell lysate at 20 µg

Lane 6:

A20 (mouse reticum sarcoma B lymphocyte) 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 1/100000 dilution

Observed band size: 42 kDa,36 kDa

false

• WB

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Western blot - Anti-CD40 antibody [RM1193] (AB317586)

Anti-CD40 antibody (ab317586) staining at 1/1000 dilution is specific to human and mouse species. Anti-CD40 antibody (ab13545) at 1/1000 dilution is weak than ab317586 in mouse tissue lysate.

The molecular weight observed is consistent with the literature (PMID : 23404288).

The lysis conditions of the tissues could be further optimised to improve performance.

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

All lanes:

Western blot - Anti-CD40 antibody [RM1193] (ab317586) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

Mouse thymus tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue 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 1/100000 dilution

Observed band size: 42 kDa,36 kDa

true

Exposure time: 136s